Engineering of systems, methods and optimized guide compositions for sequence manipulation

ABSTRACT

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR-Cas system.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application in a continuation of International Application No.PCT/US2013/074819 filed Dec. 12, 2013, and published as PCT PublicationNo. WO 2014/093712 on Jun. 19, 2014 and which claims priority to U.S.provisional patent application 61/836,127 entitled ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Jun. 17, 2013. This application also claims priority to USprovisional patent applications 61/758,468; 61/769,046; 61/802,174;61/806,375; 61/814,263; 61/819,803 and 61/828,130 each entitledENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FORSEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15,2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013respectively. Priority is also claimed to US provisional patentapplications 61/736,527 and 61/748,427, both entitled SYSTEMS METHODSAND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 andJan. 2, 2013, respectively. Priority is also claimed to US provisionalpatent applications 61/791,409 and 61/835,931 both entitledBI-2011/008/44790.02.2003 and BI-2011/008/44790.03.2003 filed on Mar.15, 2013 and Jun. 17, 2013 respectively.

Reference is also made to US provisional patent applications 61/835,936,61/836,101, 61/836,080, 61/836,123 and 61/835,973 each filed Jun. 17,2013.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support awarded by the NationalInstitutes of Health, NIH Pioneer Award DP1MH100706. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as genome perturbation or gene-editing, that may usevector systems related to Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that are affordable, easyto set up, scalable, and amenable to targeting multiple positions withinthe eukaryotic genome.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for sequence targeting with a wide array of applications.This invention addresses this need and provides related advantages. TheCRISPR/Cas or the CRISPR-Cas system (both terms are used interchangeablythroughout this application) does not require the generation ofcustomized proteins to target specific sequences but rather a single Casenzyme can be programmed by a short RNA molecule to recognize a specificDNA target, in other words the Cas enzyme can be recruited to a specificDNA target using said short RNA molecule. Adding the CRISPR-Cas systemto the repertoire of genome sequencing techniques and analysis methodsmay significantly simplify the methodology and accelerate the ability tocatalog and map genetic factors associated with a diverse range ofbiological functions and diseases. To utilize the CRISPR-Cas systemeffectively for genome editing without deleterious effects, it iscritical to understand aspects of engineering and optimization of thesegenome engineering tools, which are aspects of the claimed invention.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a tracr mate sequence and one ormore insertion sites for inserting one or more guide sequences upstreamof the tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a cell, e.g., eukaryotic cell, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracr sequence; and (b) a second regulatory elementoperably linked to an enzyme-coding sequence encoding said CRISPR enzymecomprising a nuclear localization sequence; wherein components (a) and(b) are located on the same or different vectors of the system. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a differenttarget sequence in a eukaryotic cell. In some embodiments, the systemcomprises the tracr sequence under the control of a third regulatoryelement, such as a polymerase III promoter. In some embodiments, thetracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofsequence complementarity along the length of the tracr mate sequencewhen optimally aligned. In some embodiments, the CRISPR complexcomprises one or more nuclear localization sequences of sufficientstrength to drive accumulation of said CRISPR complex in a detectableamount in the nucleus of a eukaryotic cell. Without wishing to be boundby theory, it is believed that a nuclear localization sequence is notnecessary for CRISPR complex activity in eukaryotes, but that includingsuch sequences enhances activity of the system, especially as totargeting nucleic acid molecules in the nucleus. In some embodiments,the CRISPR enzyme is a type II CRISPR system enzyme. In someembodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments,the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9,and may include mutated Cas9 derived from these organisms. The enzymemay be a Cas9 homolog or ortholog. In some embodiments, the CRISPRenzyme is codon-optimized for expression in a eukaryotic cell. In someembodiments, the CRISPR enzyme directs cleavage of one or two strands atthe location of the target sequence. In some embodiments, the CRISPRenzyme lacks DNA strand cleavage activity. In some embodiments, thefirst regulatory element is a polymerase III promoter. In someembodiments, the second regulatory element is a polymerase II promoter.In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19,20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20nucleotides in length. In general, and throughout this specification,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors.” Commonexpression vectors of utility in recombinant DNA techniques are often inthe form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g.1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters(e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the p-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF apromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit 3-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a vector comprising a regulatoryelement operably linked to an enzyme-coding sequence encoding a CRISPRenzyme comprising one or more nuclear localization sequences. In someembodiments, said regulatory element drives transcription of the CRISPRenzyme in a eukaryotic cell such that said CRISPR enzyme accumulates ina detectable amount in the nucleus of the eukaryotic cell. In someembodiments, the regulatory element is a polymerase II promoter. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. In some embodiments, the CRISPR enzyme is codon-optimized forexpression in a eukaryotic cell. In some embodiments, the CRISPR enzymedirects cleavage of one or two strands at the location of the targetsequence. In some embodiments, the CRISPR enzyme lacks DNA strandcleavage activity.

In one aspect, the invention provides a CRISPR enzyme comprising one ormore nuclear localization sequences of sufficient strength to driveaccumulation of said CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In some embodiments, the CRISPR enzyme is a typeII CRISPR system enzyme. In some embodiments, the CRISPR enzyme is aCas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S.pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derivedfrom these organisms. The enzyme may be a Cas9 homolog or ortholog. Insome embodiments, the CRISPR enzyme lacks the ability to cleave one ormore strands of a target sequence to which it binds.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a tracr mate sequenceand one or more insertion sites for inserting one or more guidesequences upstream of the tracr mate sequence, wherein when expressed,the guide sequence directs sequence-specific binding of a CRISPR complexto a target sequence in a eukaryotic cell, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracr sequence, and/or (b) a second regulatoryelement operably linked to an enzyme-coding sequence encoding saidCRISPR enzyme comprising a nuclear localization sequence. In someembodiments, the host cell comprises components (a) and (b). In someembodiments, component (a), component (b), or components (a) and (b) arestably integrated into a genome of the host eukaryotic cell. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a differenttarget sequence in a eukaryotic cell. In some embodiments, theeukaryotic host cell further comprises a third regulatory element, suchas a polymerase III promoter, operably linked to said tracr sequence. Insome embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,80%, 90%, 95%, or 99% of sequence complementarity along the length ofthe tracr mate sequence when optimally aligned. In some embodiments, theCRISPR enzyme comprises one or more nuclear localization sequences ofsufficient strength to drive accumulation of said CRISPR enzyme in adetectable amount in the nucleus of a eukaryotic cell. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. The enzyme may be a Cas9 homolog or ortholog. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence. In someembodiments, the CRISPR enzyme lacks DNA strand cleavage activity. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase 11 promoter. In some embodiments, the guide sequence is atleast 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, orbetween 15-25, or between 15-20 nucleotides in length. In an aspect, theinvention provides a non-human eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. In other aspects, theinvention provides a eukaryotic organism; preferably a multicellulareukaryotic organism, comprising a eukaryotic host cell according to anyof the described embodiments. The organism in some embodiments of theseaspects may be an animal; for example a mammal. Also, the organism maybe an arthropod such as an insect. The organism also may be a plant.Further, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a tracr mate sequence and one or more insertion sites forinserting one or more guide sequences upstream of the tracr matesequence, wherein when expressed, the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzymecomplexed with (1) the guide sequence that is hybridized to the targetsequence, and (2) the tracr mate sequence that is hybridized to thetracr sequence; and/or (b) a second regulatory element operably linkedto an enzyme-coding sequence encoding said CRISPR enzyme comprising anuclear localization sequence. In some embodiments, the kit comprisescomponents (a) and (b) located on the same or different vectors of thesystem. In some embodiments, component (a) further comprises the tracrsequence downstream of the tracr mate sequence under the control of thefirst regulatory element. In some embodiments, component (a) furthercomprises two or more guide sequences operably linked to the firstregulatory element, wherein when expressed, each of the two or moreguide sequences direct sequence specific binding of a CRISPR complex toa different target sequence in a eukaryotic cell. In some embodiments,the system further comprises a third regulatory element, such as apolymerase III promoter, operably linked to said tracr sequence. In someembodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%,90%, 95%, or 99% of sequence complementarity along the length of thetracr mate sequence when optimally aligned. In some embodiments, theCRISPR enzyme comprises one or more nuclear localization sequences ofsufficient strength to drive accumulation of said CRISPR enzyme in adetectable amount in the nucleus of a eukaryotic cell. In someembodiments, the CRISPR enzyme is a type II CRISPR system enzyme. Insome embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes or S.thermophilus Cas9, and may include mutated Cas9 derived from theseorganisms. The enzyme may be a Cas9 homolog or ortholog. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence. In someembodiments, the CRISPR enzyme lacks DNA strand cleavage activity. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase II promoter. In some embodiments, the guide sequence is atleast 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, orbetween 15-25, or between 15-20 nucleotides in length.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.In some embodiments, said cleavage comprises cleaving one or two strandsat the location of the target sequence by said CRISPR enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsto said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence. In someembodiments, said vectors are delivered to the eukaryotic cell in asubject. In some embodiments, said modifying takes place in saideukaryotic cell in a cell culture. In some embodiments, the methodfurther comprises isolating said eukaryotic cell from a subject prior tosaid modifying. In some embodiments, the method further comprisesreturning said eukaryotic cell and/or cells derived therefrom to saidsubject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence. Insome embodiments, the method further comprises delivering one or morevectors to said eukaryotic cells, wherein the one or more vectors driveexpression of one or more of: the CRISPR enzyme, the guide sequencelinked to the tracr mate sequence, and the tracr sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated with an increase in the risk ofhaving or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors into a eukaryotic cell,wherein the one or more vectors drive expression of one or more of: aCRISPR enzyme, a guide sequence linked to a tracr mate sequence, and atracr sequence; and (b) allowing a CRISPR complex to bind to a targetpolynucleotide to effect cleavage of the target polynucleotide withinsaid disease gene, wherein the CRISPR complex comprises the CRISPRenzyme complexed with (1) the guide sequence that is hybridized to thetarget sequence within the target polynucleotide, and (2) the tracr matesequence that is hybridized to the tracr sequence, thereby generating amodel eukaryotic cell comprising a mutated disease gene. In someembodiments, said cleavage comprises cleaving one or two strands at thelocation of the target sequence by said CRISPR enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpression from a gene comprising the target sequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated with an increase in the risk of having or developinga disease. In some embodiments, the method comprises (a) contacting atest compound with a model cell of any one of the described embodiments;and (b) detecting a change in a readout that is indicative of areduction or an augmentation of a cell signaling event associated withsaid mutation in said disease gene, thereby developing said biologicallyactive agent that modulates said cell signaling event associated withsaid disease gene.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence upstream of a tracr mate sequence, whereinthe guide sequence when expressed directs sequence-specific binding of aCRISPR complex to a corresponding target sequence present in aeukaryotic cell. In some embodiments, the target sequence is a viralsequence present in a eukaryotic cell. In some embodiments, the targetsequence is a proto-oncogene or an oncogene.

In one aspect the invention provides for a method of selecting one ormore prokaryotic cell(s) by introducing one or more mutations in a genein the one or more prokaryotic cell (s), the method comprising:introducing one or more vectors into the prokaryotic cell (s), whereinthe one or more vectors drive expression of one or more of: a CRISPRenzyme, a guide sequence linked to a tracr mate sequence, a tracrsequence, and a editing template; wherein the editing template comprisesthe one or more mutations that abolish CRISPR enzyme cleavage; allowinghomologous recombination of the editing template with the targetpolynucleotide in the cell(s) to be selected; allowing a CRISPR complexto bind to a target polynucleotide to effect cleavage of the targetpolynucleotide within said gene, wherein the CRISPR complex comprisesthe CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence within the target polynucleotide, and(2) the tracr mate sequence that is hybridized to the tracr sequence,wherein binding of the CRISPR complex to the target polynucleotideinduces cell death, thereby allowing one or more prokaryotic cell(s) inwhich one or more mutations have been introduced to be selected. In apreferred embodiment, the CRISPR enzyme is Cas9. In another aspect ofthe invention the cell to be selected may be a eukaryotic cell. Aspectsof the invention allow for selection of specific cells without requiringa selection marker or a two-step process that may include acounter-selection system.

In some aspects the invention provides a non-naturally occurring orengineered composition comprising a CRISPR-Cas system chimeric RNA(chiRNA) polynucleotide sequence, wherein the polynucleotide sequencecomprises (a) a guide sequence capable of hybridizing to a targetsequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) atracr sequence wherein (a), (b) and (c) are arranged in a 5′ to 3′orientation, wherein when transcribed, the tracr mate sequencehybridizes to the tracr sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1)the guide sequence that is hybridized to the target sequence, and (2)the tracr mate sequence that is hybridized to the tracr sequence,

or

a CRISPR enzyme system, wherein the system is encoded by a vector systemcomprising one or more vectors comprising I. a first regulatory elementoperably linked to a CRISPR-Cas system chimeric RNA (chiRNA)polynucleotide sequence, wherein the polynucleotide sequence comprises(a) one or more guide sequences capable of hybridizing to one or moretarget sequences in a eukaryotic cell, (b) a tracr mate sequence, and(c) one or more tracr sequences, and II. a second regulatory elementoperably linked to an enzyme-coding sequence encoding a CRISPR enzymecomprising at least one or more nuclear localization sequences, wherein(a), (b) and (c) are arranged in a 5′ to 3′orientation, whereincomponents I and II are located on the same or different vectors of thesystem, wherein when transcribed, the tracr mate sequence hybridizes tothe tracr sequence and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, wherein the CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the guidesequence that is hybridized to the target sequence, and (2) the tracrmate sequence that is hybridized to the tracr sequence, or a multiplexedCRISPR enzyme system, wherein the system is encoded by a vector systemcomprising one or more vectors comprising I. a first regulatory elementoperably linked to (a) one or more guide sequences capable ofhybridizing to a target sequence in a cell, and (b) at least one or moretracr mate sequences, II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme, and III. a thirdregulatory element operably linked to a tracr sequence, whereincomponents I, II and III are located on the same or different vectors ofthe system, wherein when transcribed, the tracr mate sequence hybridizesto the tracr sequence and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, wherein the CRISPRcomplex comprises the CRISPR enzyme complexed with (1) the guidesequence that is hybridized to the target sequence, and (2) the tracrmate sequence that is hybridized to the tracr sequence, and wherein inthe multiplexed system multiple guide sequences and a single tracrsequence is used; and wherein one or more of the guide, tracr and tracrmate sequences are modified to improve stability.

In aspects of the invention, the modification comprises an engineeredsecondary structure. For example, the modification can comprise areduction in a region of hybridization between the tracr mate sequenceand the tracr sequence. For example, the modification also may comprisefusing the tracr mate sequence and the tracr sequence through anartificial loop. The modification may comprise the tracr sequence havinga length between 40 and 120 bp. In embodiments of the invention, thetracr sequence is between 40 bp and full length of the tracr. In certainembodiments, the length of tracRNA includes at least nucleotides 1-67and in some embodiments at least nucleotides 1-85 of the wild typetracRNA. In some embodiments, at least nucleotides corresponding tonucleotides 1-67 or 1-85 of wild type S. pyogenes Cas9 tracRNA may beused. Where the CRISPR system uses enzymes other than Cas9, or otherthan SpCas9, then corresponding nucleotides in the relevant wild typetracRNA may be present. In some embodiments, the length of tracRNAincludes no more than nucleotides 1-67 or 1-85 of the wild type tracRNA.The modification may comprise sequence optimization. In certain aspects,sequence optimization may comprise reducing the incidence of polyTsequences in the tracr and/or tracr mate sequence. Sequence optimizationmay be combined with reduction in the region of hybridization betweenthe tracr mate sequence and the tracr sequence; for example, a reducedlength tracr sequence.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the modification comprises reduction in polyTsequences in the tracr and/or tracr mate sequence. In some aspects ofthe invention, one or more Ts present in a poly-T sequence of therelevant wild type sequence (that is, a stretch of more than 3, 4, 5, 6,or more contiguous T bases; in some embodiments, a stretch of no morethan 10, 9, 8, 7, 6 contiguous T bases) may be substituted with a non-Tnucleotide, e.g., an A, so that the string is broken down into smallerstretches of Ts with each stretch having 4, or fewer than 4 (forexample, 3 or 2) contiguous Ts. Bases other than A may be used forsubstitution, for example C or G, or non-naturally occurring nucleotidesor modified nucleotides. If the string of Ts is involved in theformation of a hairpin (or stem loop), then it is advantageous that thecomplementary base for the non-T base be changed to complement the non-Tnucleotide. For example, if the non-T base is an A, then its complementmay be changed to a T, e.g., to preserve or assist in the preservationof secondary structure. For instance, 5′-TTTTT can be altered to become5′-TTTAT and the complementary 5′-AAAAA can be changed into 5′-ATAAA.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the modification comprises adding a polyTterminator sequence. In an aspect the invention provides the CRISPR-Cassystem or CRISPR enzyme system wherein the modification comprises addinga polyT terminator sequence in tracr and/or tracr mate sequences. In anaspect the invention provides the CRISPR-Cas system or CRISPR enzymesystem wherein the modification comprises adding a polyT terminatorsequence in the guide sequence. The polyT terminator sequence maycomprise 5 contiguous T bases, or more than 5.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the modification comprises altering loops and/orhairpins. In an aspect the invention provides the CRISPR-Cas system orCRISPR enzyme system wherein the modification comprises providing aminimum of two hairpins in the guide sequence. In an aspect theinvention provides the CRISPR-Cas system or CRISPR enzyme system whereinthe modification comprises providing a hairpin formed by complementationbetween the tracr and tracr mate (direct repeat) sequence. In an aspectthe invention provides the CRISPR-Cas system or CRISPR enzyme systemwherein the modification comprises providing one or more furtherhairpin(s) at or towards the 3′ end of the tracrRNA sequence. Forexample, a hairpin may be formed by providing self complementarysequences within the tracRNA sequence joined by a loop such that ahairpin is formed on self folding. In an aspect the invention providesthe CRISPR-Cas system or CRISPR enzyme system wherein the modificationcomprises providing additional hairpins added to the 3′ of the guidesequence. In an aspect the invention provides the CRISPR-Cas system orCRISPR enzyme system wherein the modification comprises extending the 5′end of the guide sequence. In an aspect the invention provides theCRISPR-Cas system or CRISPR enzyme system wherein the modificationcomprises providing one or more hairpins in the 5′ end of the guidesequence. In an aspect the invention provides the CRISPR-Cas system orCRISPR enzyme system wherein the modification comprises appending thesequence (5′-AGGACGAAGTCCTAA) to the 5′ end of the guide sequence. Othersequences suitable for forming hairpins will be known to the skilledperson, and may be used in certain aspects of the invention. In someaspects of the invention, at least 2, 3, 4, 5, or more additionalhairpins are provided. In some aspects of the invention, no more than10, 9, 8, 7, 6 additional hairpins are provided. In an aspect theinvention provides the CRISPR-Cas system or CRISPR enzyme system whereinthe modification comprises two hairpins. In an aspect the inventionprovides the CRISPR-Cas system or CRISPR enzyme system wherein themodification comprises three hairpins. In an aspect the inventionprovides the CRISPR-Cas system or CRISPR enzyme system wherein themodification comprises at most five hairpins.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the modification comprises providing crosslinking, or providing one or more modified nucleotides in thepolynucleotide sequence. Modified nucleotides and/or cross linking maybe provided in any or all of the tracr, tracr mate, and/or guidesequences, and/or in the enzyme coding sequence, and/or in vectorsequences. Modifications may include inclusion of at least one nonnaturally occurring nucleotide, or a modified nucleotide, or analogsthereof. Modified nucleotides may be modified at the ribose, phosphate,and/or base moiety. Modified nucleotides may include 2′-O-methylanalogs, 2′-deoxy analogs, or 2′-fluoro analogs. The nucleic acidbackbone may be modified, for example, a phosphorothioate backbone maybe used. The use of locked nucleic acids (LNA) or bridged nucleic acids(BNA) may also be possible. Further examples of modified bases include,but are not limited to. 2-aminopurine, 5-bromo-uridine, pseudouridine,inosine, 7-methylguanosine.

It will be understood that any or all of the above modifications may beprovided in isolation or in combination in a given CRISPR-Cas system orCRISPR enzyme system. Such a system may include one, two, three, four,five, or more of said modifications.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the CRISPR enzyme is a type II CRISPR systemenzyme, e.g., a Cas9 enzyme. In an aspect the invention provides theCRISPR-Cas system or CRISPR enzyme system wherein the CRISPR enzyme iscomprised of less than one thousand amino acids, or less than fourthousand amino acids. In an aspect the invention provides the CRISPR-Cassystem or CRISPR enzyme system wherein the Cas9 enzyme is StCas9 orSt1Cas9, or the Cas9 enzyme is a Cas9 enzyme from an organism selectedfrom the group consisting of genus Streptococcus, Campylobacter,Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria,Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus,Eubacterium or Corynebacter. In an aspect the invention provides theCRISPR-Cas system or CRISPR enzyme system wherein the CRISPR enzyme is anuclease directing cleavage of both strands at the location of thetarget sequence.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the first regulatory element is a polymerase IIIpromoter. In an aspect the invention provides the CRISPR-Cas system orCRISPR enzyme system wherein the second regulatory element is apolymerase II promoter.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the guide sequence comprises at least fifteennucleotides.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the modification comprises optimized tracrsequence and/or optimized guide sequence RNA and/or co-fold structure oftracr sequence and/or tracr mate sequence(s) and/or stabilizingsecondary structures of tracr sequence and/or tracr sequence with areduced region of base-pairing and/or tracr sequence fused RNA elements;and/or, in the multiplexed system there are two RNAs comprising a tracerand comprising a plurality of guides or one RNA comprising a pluralityof chimerics.

In aspects of the invention the chimeric RNA architecture is furtheroptimized according to the results of mutagenesis studies. In chimericRNA with two or more hairpins, mutations in the proximal direct repeatto stabilize the hairpin may result in ablation of CRISPR complexactivity. Mutations in the distal direct repeat to shorten or stabilizethe hairpin may have no effect on CRISPR complex activity. Sequencerandomization in the bulge region between the proximal and distalrepeats may significantly reduce CRISPR complex activity. Single basepair changes or sequence randomization in the linker region betweenhairpins may result in complete loss of CRISPR complex activity. Hairpinstabilization of the distal hairpins that follow the first hairpin afterthe guide sequence may result in maintenance or improvement of CRISPRcomplex activity. Accordingly, in preferred embodiments of theinvention, the chimeric RNA architecture may be further optimized bygenerating a smaller chimeric RNA which may be beneficial fortherapeutic delivery options and other uses and this may be achieved byaltering the distal direct repeat so as to shorten or stabilize thehairpin. In further preferred embodiments of the invention, the chimericRNA architecture may be further optimized by stabilizing one or more ofthe distal hairpins. Stabilization of hairpins may include modifyingsequences suitable for forming hairpins. In some aspects of theinvention, at least 2, 3, 4, 5, or more additional hairpins areprovided. In some aspects of the invention, no more than 10, 9, 8, 7, 6additional hairpins are provided. In some aspects of the inventionstabilization may be cross linking and other modifications.Modifications may include inclusion of at least one non naturallyoccurring nucleotide, or a modified nucleotide, or analogs thereof.Modified nucleotides may be modified at the ribose, phosphate, and/orbase moiety. Modified nucleotides may include 2′-O-methyl analogs,2′-deoxy analogs, or 2′-fluoro analogs. The nucleic acid backbone may bemodified, for example, a phosphorothioate backbone may be used. The useof locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also bepossible. Further examples of modified bases include, but are notlimited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine,7-methylguanosine.

In an aspect the invention provides the CRISPR-Cas system or CRISPRenzyme system wherein the CRISPR enzyme is codon-optimized forexpression in a eukaryotic cell.

Accordingly, in some aspects of the invention, the length of tracRNArequired in a construct of the invention, e.g., a chimeric construct,need not necessarily be fixed, and in some aspects of the invention itcan be between 40 and 120 bp, and in some aspects of the invention up tothe full length of the tracr, e.g., in some aspects of the invention,until the 3′ end of tracr as punctuated by the transcription terminationsignal in the bacterial genome. In certain embodiments, the length oftracRNA includes at least nucleotides 1-67 and in some embodiments atleast nucleotides 1-85 of the wild type tracRNA. In some embodiments, atleast nucleotides corresponding to nucleotides 1-67 or 1-85 of wild typeS. pyogenes Cas9 tracRNA may be used. Where the CRISPR system usesenzymes other than Cas9, or other than SpCas9, then correspondingnucleotides in the relevant wild type tracRNA may be present. In someembodiments, the length of tracRNA includes no more than nucleotides1-67 or 1-85 of the wild type tracRNA With respect to sequenceoptimization (e.g., reduction in polyT sequences), e.g., as to stringsof Ts internal to the tracr mate (direct repeat) or tracrRNA, in someaspects of the invention, one or more Ts present in a poly-T sequence ofthe relevant wild type sequence (that is, a stretch of more than 3, 4,5, 6, or more contiguous T bases; in some embodiments, a stretch of nomore than 10, 9, 8, 7, 6 contiguous T bases) may be substituted with anon-T nucleotide, e.g., an A, so that the string is broken down intosmaller stretches of Ts with each stretch having 4, or fewer than 4 (forexample, 3 or 2) contiguous Ts. If the string of Ts is involved in theformation of a hairpin (or stem loop), then it is advantageous that thecomplementary base for the non-T base be changed to complement the non-Tnucleotide. For example, if the non-T base is an A, then its complementmay be changed to a T, e.g., to preserve or assist in the preservationof secondary structure. For instance, 5′-TTTTT can be altered to become5′-TTTAT and the complementary 5′-AAAAA can be changed into 5′-ATAAA. Asto the presence of polyT terminator sequences in tracr+tracr matetranscript, e.g., a polyT terminator (TTTTT or more), in some aspects ofthe invention it is advantageous that such be added to end of thetranscript, whether it is in two RNA (tracr and tracr mate) or singleguide RNA form. Concerning loops and hairpins in tracr and tracr matetranscripts, in some aspects of the invention it is advantageous that aminimum of two hairpins be present in the chimeric guide RNA. A firsthairpin can be the hairpin formed by complementation between the tracrand tracr mate (direct repeat) sequence. A second hairpin can be at the3′ end of the tracrRNA sequence, and this can provide secondarystructure for interaction with Cas9. Additional hairpins may be added tothe 3′ of the guide RNA, e.g., in some aspects of the invention toincrease the stability of the guide RNA. Additionally, the 5′ end of theguide RNA, in some aspects of the invention, may be extended. In someaspects of the invention, one may consider 20 bp in the 5′ end as aguide sequence. The 5′ portion may be extended. One or more hairpins canbe provided in the 5′ portion, e.g., in some aspects of the invention,this may also improve the stability of the guide RNA. In some aspects ofthe invention, the specific hairpin can be provided by appending thesequence (5′-AGGACGAAGTCCTAA) to the 5′ end of the guide sequence, and,in some aspects of the invention, this may help improve stability. Othersequences suitable for forming hairpins will be known to the skilledperson, and may be used in certain aspects of the invention. In someaspects of the invention, at least 2, 3, 4, 5, or more additionalhairpins are provided. In some aspects of the invention, no more than10, 9, 8, 7, 6 additional hairpins are provided. The foregoing alsoprovides aspects of the invention involving secondary structure in guidesequences. In some aspects of the invention there may be cross linkingand other modifications, e.g., to improve stability. Modifications mayinclude inclusion of at least one non naturally occurring nucleotide, ora modified nucleotide, or analogs thereof. Modified nucleotides may bemodified at the ribose, phosphate, and/or base moiety. Modifiednucleotides may include 2′-O-methyl analogs, 2′-deoxy analogs, or2′-fluoro analogs. The nucleic acid backbone may be modified, forexample, a phosphorothioate backbone may be used. The use of lockednucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible.Further examples of modified bases include, but are not limited to,2-aminopurine, 5-bromo-uridine, pseudouridine, inosine,7-methylguanosine. Such modifications or cross linking may be present inthe guide sequence or other sequences adjacent the guide sequence.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”. “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. These and other embodiments aredisclosed or are obvious from and encompassed by, the following DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a schematic model of the CRISPR system. The Cas9 nucleasefrom Streptococcus pyogenes (yellow) is targeted to genomic DNA by asynthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue)and a scaffold (red). The guide sequence base-pairs with the DNA target(blue), directly upstream of a requisite 5′-NGG protospacer adjacentmotif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ˜3bp upstream of the PAM (red triangle).

FIG. 2A-F illustrates an exemplary CRISPR system, a possible mechanismof action, an example adaptation for expression in eukaryotic cells, andresults of tests assessing nuclear localization and CRISPR activity.

FIG. 3A-C illustrates an exemplary expression cassette for expression ofCRISPR system elements in eukaryotic cells, predicted structures ofexample guide sequences, and CRISPR system activity as measured ineukaryotic and prokaryotic cells.

FIG. 4A-D illustrates results of an evaluation of SpCas9 specificity foran example target.

FIG. 5A-G illustrates an exemplary vector system and results for its usein directing homologous recombination in eukaryotic cells.

FIG. 6A-C illustrates a comparison of different tracrRNA transcripts forCas9-mediated gene targeting.

FIG. 7A-D illustrates an exemplary CRISPR system, an example adaptationfor expression in eukaryotic cells, and results of tests assessingCRISPR activity.

FIG. 8A-C illustrates exemplary manipulations of a CRISPR system fortargeting of genomic loci in mammalian cells.

FIG. 9A-B illustrates the results of a Northern blot analysis of crRNAprocessing in mammalian cells.

FIG. 10A-C illustrates a schematic representation of chimeric RNAs andresults of SURVEYOR assays for CRISPR system activity in eukaryoticcells.

FIG. 11A-B illustrates a graphical representation of the results ofSURVEYOR assays for CRISPR system activity in eukaryotic cells.

FIG. 12 illustrates predicted secondary structures for exemplarychimeric RNAs comprising a guide sequence, tracr mate sequence, andtracr sequence.

FIG. 13A-D is a phylogenetic tree of Cas genes

FIG. 14A-F shows the phylogenetic analysis revealing five families ofCas9s, including three groups of large Cas9s (˜1400 amino acids) and twoof small Cas9s (˜1100 amino acids).

FIG. 15 shows a graph depicting the function of different optimizedguide RNAs.

FIG. 16 shows the sequence and structure of different guide chimericRNAs.

FIG. 17 shows the co-fold structure of the tracrRNA and direct repeat.

FIGS. 18 A and B shows data from the StlCas9 chimeric guide RNAoptimization in vitro.

FIG. 19A-B shows cleavage of either unmethylated or methylated targetsby SpCas9 cell lysate.

FIG. 20A-G shows the optimization of guide RNA architecture forSpCas9-mediated mammalian genome editing. (a) Schematic of bicistronicexpression vector (PX330) for U6 promoter-driven single guide RNA(sgRNA) and CBh promoter-driven human codon-optimized Streptococcuspyogenes Cas9 (hSpCas9) used for all subsequent experiments. The sgRNAconsists of a 20-nt guide sequence (blue) and scaffold (red), truncatedat various positions as indicated. (b) SURVEYOR assay forSpCas9-mediated indels at the human EMX1 and PVALB loci. Arrows indicatethe expected SURVEYOR fragments (n=3). (c) Northern blot analysis forthe four sgRNA truncation architectures, with U1 as loading control. (d)Both wildtype (wt) or nickase mutant (D10A) of SpCas9 promoted insertionof a HindIII site into the human EMX1 gene. Single strandedoligonucleotides (ssODNs), oriented in either the sense or antisensedirection relative to genome sequence, were used as homologousrecombination templates. (e) Schematic of the human SERPINB5 locus.sgRNAs and PAMs are indicated by colored bars above sequence;methylcytosine (Me) are highlighted (pink) and numbered relative to thetranscriptional start site (TSS, +1). (f) Methylation status of SERPINB5assayed by bisulfite sequencing of 16 clones. Filled circles, methylatedCpG; open circles, unmethylated CpG. (g) Modification efficiency bythree sgRNAs targeting the methylated region of SERPINB5, assayed bydeep sequencing (n=2). Error bars indicate Wilson intervals (OnlineMethods).

FIG. 21A-B shows the further optimization of CRISPR-Cas sgRNAarchitecture. (a) Schematic of four additional sgRNA architectures,I-IV. Each consists of a 20-nt guide sequence (blue) joined to thedirect repeat (DR, grey), which hybridizes to the tracrRNA (red). TheDR-tracrRNA hybrid is truncated at +12 or +22, as indicated, with anartificial GAAA stem loop. tracrRNA truncation positions are numberedaccording to the previously reported transcription start site fortracrRNA. sgRNA architectures II and IV carry mutations within theirpoly-U tracts, which could serve as premature transcriptionalterminators. (b) SURVEYOR assay for SpCas9-mediated indels at the humanEMX1 locus for target sites 1-3. Arrows indicate the expected SURVEYORfragments (n=3).

FIG. 22 illustrates visualization of some target sites in the humangenome.

FIG. 23A-B shows (A) a schematic of the sgRNA and (B) the SURVEYORanalysis of five sgRNA variants for SaCas9 for an optimal truncatedarchitecture with highest cleavage efficiency

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide maycomprise one or more modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component.

In aspects of the invention the terms “chimeric RNA”, “chimeric guideRNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are usedinterchangeably and refer to the polynucleotide sequence comprising theguide sequence, the tracr sequence and the tracr mate sequence. The term“guide sequence” refers to the about 20 bp sequence within the guide RNAthat specifies the target site and may be used interchangeably with theterms “guide” or “spacer”. The term “tracr mate sequence” may also beused interchangeably with the term “direct repeat(s)”.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick base-pairing or other non-traditional types. Apercent complementarity indicates the percentage of residues in anucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

As used herein, “stabilization” or “increasing stability” with respectto components of the CRISPR system relate to securing or steadying thestructure of the molecule. This may be accomplished by introduction ofone or mutations, including single or multiple base pair changes,increasing the number of hair pins, cross linking, breaking upparticular stretches of nucleotides and other modifications.Modifications may include inclusion of at least one non naturallyoccurring nucleotide, or a modified nucleotide, or analogs thereof.Modified nucleotides may be modified at the ribose, phosphate, and/orbase moiety. Modified nucleotides may include 2′-O-methyl analogs,2′-deoxy analogs, or 2′-fluoro analogs. The nucleic acid backbone may bemodified, for example, a phosphorothioate backbone may be used. The useof locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also bepossible. Further examples of modified bases include, but are notlimited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine,7-methylguanosine. These modifications may apply to any component of theCRSIPR system. In a preferred embodiment these modifications are made tothe RNA components, e.g. the guide RNA or chimeric polynucleotidesequence.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

The terms “subject.” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed. In some embodiments, asubject may be an invertebrate animal, for example, an insect or anematode; while in others, a subject may be a plant or a fungus.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press. San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Vectors may be introduced and propagated in a prokaryote. In someembodiments, a prokaryote is used to amplify copies of a vector to beintroduced into a eukaryotic cell or as an intermediate vector in theproduction of a vector to be introduced into a eukaryotic cell (e.g.amplifying a plasmid as part of a viral vector packaging system). Insome embodiments, a prokaryote is used to amplify copies of a vector andexpress one or more nucleic acids, such as to provide a source of one ormore proteins for delivery to a host cell or host organism. Expressionof proteins in prokaryotes is most often carried out in Escherichia coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, such as to theamino terminus of the recombinant protein. Such fusion vectors may serveone or more purposes, such as: (i) to increase expression of recombinantprotein; (ii) to increase the solubility of the recombinant protein; and(iii) to aid in the purification of the recombinant protein by acting asa ligand in affinity purification. Often, in fusion expression vectors,a proteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacteriumn tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. In some embodiments, oneor more elements of a CRISPR system is derived from a type I, type II,or type III CRISPR system. In some embodiments, one or more elements ofa CRISPR system is derived from a particular organism comprising anendogenous CRISPR system, such as Streptococcus pyogenes. In general, aCRISPR system is characterized by elements that promote the formation ofa CRISPR complex at the site of a target sequence (also referred to as aprotospacer in the context of an endogenous CRISPR system). In thecontext of formation of a CRISPR complex, “target sequence” refers to asequence to which a guide sequence is designed to have complementarity,where hybridization between a target sequence and a guide sequencepromotes the formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridisation and promote formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, the targetsequence may be within an organelle of a eukaryotic cell, for example,mitochondrion or chloroplast. A sequence or template that may be usedfor recombination into the the targeted locus comprising the targetsequences is referred to as an “editing template” or “editingpolynucleotide” or “editing sequence”. In aspects of the invention, anexogenous template polynucleotide may be referred to as an editingtemplate. In an aspect of the invention the recombination is homologousrecombination.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tracr sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence. In some embodiments, the tracrsequence has sufficient complementarity to a tracr mate sequence tohybridise and participate in formation of a CRISPR complex. As with thetarget sequence, it is believed that complete complementarity is notneeded, provided there is sufficient to be functional. In someembodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%,95% or 99% of sequence complementarity along the length of the tracrmate sequence when optimally aligned. In some embodiments, one or morevectors driving expression of one or more elements of a CRISPR systemare introduced into a host cell such that expression of the elements ofthe CRISPR system direct formation of a CRISPR complex at one or moretarget sites. For example, a Cas enzyme, a guide sequence linked to atracr-mate sequence, and a tracr sequence could each be operably linkedto separate regulatory elements on separate vectors. Alternatively, twoor more of the elements expressed from the same or different regulatoryelements, may be combined in a single vector, with one or moreadditional vectors providing any components of the CRISPR system notincluded in the first vector. CRISPR system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a CRISPR enzyme and one or more of the guidesequence, tracr mate sequence (optionally operably linked to the guidesequence), and a tracr sequence embedded within one or more intronsequences (e.g. each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the CRISPRenzyme, guide sequence, tracr mate sequence, and tracr sequence areoperably linked to and expressed from the same promoter.

In some embodiments, a vector comprises one or more insertion sites,such as a restriction endonuclease recognition sequence (also referredto as a “cloning site”). In some embodiments, one or more insertionsites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore insertion sites) are located upstream and/or downstream of one ormore sequence elements of one or more vectors. In some embodiments, avector comprises an insertion site upstream of a tracr mate sequence,and optionally downstream of a regulatory element operably linked to thetracr mate sequence, such that following insertion of a guide sequenceinto the insertion site and upon expression the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell. In some embodiments, a vector comprises two or moreinsertion sites, each insertion site being located between two tracrmate sequences so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target CRISPR activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell.

In some embodiments, a vector comprises a regulatory element operablylinked to an enzyme-coding sequence encoding a CRISPR enzyme, such as aCas protein. Non-limiting examples of Cas proteins include Cas1, CasB,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologs thereof, or modified versions thereof. Theseenzymes are known; for example, the amino acid sequence of S. pyogenesCas9 protein may be found in the SwissProt database under accessionnumber Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNAcleavage activity, such as Cas9. In some embodiments the CRISPR enzymeis Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandsat the location of a target sequence, such as within the target sequenceand/or within the complement of the target sequence. In someembodiments, the CRISPR enzyme directs cleavage of one or both strandswithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200,500, or more base pairs from the first or last nucleotide of a targetsequence. In some embodiments, a vector encodes a CRISPR enzyme that ismutated to with respect to a corresponding wild-type enzyme such thatthe mutated CRISPR enzyme lacks the ability to cleave one or bothstrands of a target polynucleotide containing a target sequence. Forexample, an aspartate-to-alanine substitution (D10A) in the RuvC Icatalytic domain of Cas9 from S. pyogenes converts Cas9 from a nucleasethat cleaves both strands to a nickase (cleaves a single strand). Otherexamples of mutations that render Cas9 a nickase include, withoutlimitation, H840A, N854A, and N863A. In some embodiments, a Cas9 nickasemay be used in combination with guide sequenc(es), e.g., two guidesequences, which target respectively sense and antisense strands of theDNA target. This combination allows both strands to be nicked and usedto induce NHEJ. Applicants have demonstrated (data not shown) theefficacy of two nickase targets (i.e., sgRNAs targeted at the samelocation but to different strands of DNA) in inducing mutagenic NHEJ. Asingle nickase (Cas9-D10A with a single sgRNA) is unable to induce NHEJand create indels but Applicants have shown that double nickase(Cas9-D10A and two sgRNAs targeted to different strands at the samelocation) can do so in human embryonic stem cells (hESCs). Theefficiency is about 50% of nuclease (i.e., regular Cas9 without D10mutation) in hESCs.

As a further example, two or more catalytic domains of Cas9 (RuvC I,RuvC II, and RuvC III) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. In some embodiments, aD10A mutation is combined with one or more of H840A, N854A, or N863Amutations to produce a Cas9 enzyme substantially lacking all DNAcleavage activity. In some embodiments, a CRISPR enzyme is considered tosubstantially lack all DNA cleavage activity when the DNA cleavageactivity of the mutated enzyme is less than about 25%, 10%, 5%, 1%,0.1%, 0.01%, or lower with respect to its non-mutated form. Othermutations may be useful; where the Cas9 or other CRISPR enzyme is from aspecies other than S. pyogenes, mutations in corresponding amino acidsmay be made to achieve similar effects.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzymeis codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human primate. In general, codonoptimization refers to a process of modifying a nucleic acid sequencefor enhanced expression in the host cells of interest by replacing atleast one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15,20, 25, 50, or more codons) of the native sequence with codons that aremore frequently or most frequently used in the genes of that host cellwhile maintaining the native amino acid sequence. Various speciesexhibit particular bias for certain codons of a particular amino acid.Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database”, and these tables can be adapted in a number of ways.See Nakamura, Y., et al. “Codon usage tabulated from the internationalDNA sequence databases: status for the year 2000” Nucl. Acids Res.28:292 (2000). Computer algorithms for codon optimizing a particularsequence for expression in a particular host cell are also available,such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In someembodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50,or more, or all codons) in a sequence encoding a CRISPR enzymecorrespond to the most frequently used codon for a particular aminoacid.

In some embodiments, a vector encodes a CRISPR enzyme comprising one ormore nuclear localization sequences (NLSs), such as about or more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments,the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near thecarboxy-terminus, or a combination of these (e.g. one or more NLS at theamino-terminus and one or more NLS at the carboxy terminus). When morethan one NLS is present, each may be selected independently of theothers, such that a single NLS may be present in more than one copyand/or in combination with one or more other NLSs present in one or morecopies. In a preferred embodiment of the invention, the CRISPR enzymecomprises at most 6 NLSs. In some embodiments, an NLS is considered nearthe N- or C-terminus when the nearest amino acid of the NLS is withinabout 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acidsalong the polypeptide chain from the N- or C-terminus. Typically, an NLSconsists of one or more short sequences of positively charged lysines orarginines exposed on the protein surface, but other types of NLS areknown. Non-limiting examples of NLSs include an NLS sequence derivedfrom: the NLS of the SV40 virus large T-antigen, having the amino acidsequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasminbipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS havingthe amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLShaving the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain fromimportin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma Tprotein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAPof mouse c-ab1 IV; the sequences DRLRR and PKQKKRK of the influenzavirus NS1; the sequence RKLKKKIKKL of the Hepatitis virus delta antigen;the sequence REKKKFLKRR of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK of the human poly(ADP-ribose) polymerase; and thesequence RKCLQAGMNLEARKTKK of the steroid hormone receptors (human)glucocorticoid.

In general, the one or more NLSs are of sufficient strength to driveaccumulation of the CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In general, strength of nuclear localizationactivity may derive from the number of NLSs in the CRISPR enzyme, theparticular NLS(s) used, or a combination of these factors. Detection ofaccumulation in the nucleus may be performed by any suitable technique.For example, a detectable marker may be fused to the CRISPR enzyme, suchthat location within a cell may be visualized, such as in combinationwith a means for detecting the location of the nucleus (e.g. a stainspecific for the nucleus such as DAPI). Examples of detectable markersinclude fluorescent proteins (such as Green fluorescent proteins, orGFP; RFP; CFP), and epitope tags (HA tag, flag tag, SNAP tag). Cellnuclei may also be isolated from cells, the contents of which may thenbe analyzed by any suitable process for detecting protein, such asimmunohistochemistry, Western blot, or enzyme activity assay.Accumulation in the nucleus may also be determined indirectly, such asby an assay for the effect of CRISPR complex formation (e.g. assay forDNA cleavage or mutation at the target sequence, or assay for alteredgene expression activity affected by CRISPR complex formation and/orCRISPR enzyme activity), as compared to a control no exposed to theCRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the oneor more NLSs.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about ormore than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In some embodiments, a guide sequence is less than about 75,50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Theability of a guide sequence to direct sequence-specific binding of aCRISPR complex to a target sequence may be assessed by any suitableassay. For example, the components of a CRISPR system sufficient to forma CRISPR complex, including the guide sequence to be tested, may beprovided to a host cell having the corresponding target sequence, suchas by transfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay as described herein.Similarly, cleavage of a target polynucleotide sequence may be evaluatedin a test tube by providing the target sequence, components of a CRISPRcomplex, including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome. For example, for the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A, G, T, or C; and Xcan be anything) has a single occurrence in the genome. A unique targetsequence in a genome may include an S. pyogenes Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C;and X can be anything) has a single occurrence in the genome. For the S.thermophilus CRISPR1 Cas9, a unique target sequence in a genome mayinclude a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW whereNNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is Aor T) has a single occurrence in the genome. A unique target sequence ina genome may include an S. thermophilus CRISPR1 Cas9 target site of theform MMMMMMMMMNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNXXAGAAW (N is A, G, T,or C; X can be anything; and W is A or T) has a single occurrence in thegenome. For the S. pyogenes Cas9, a unique target sequence in a genomemay include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXGwhere NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) hasa single occurrence in the genome. A unique target sequence in a genomemay include an S. pyogenes Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG (N is A, G, T, or C;and X can be anything) has a single occurrence in the genome. In each ofthese sequences “M” may be A, G, T, or C, and need not be considered inidentifying a sequence as unique.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. TBA (Broad Reference BI-2012/084 44790.11.2022); incorporatedherein by reference.

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa CRISPR complex at a target sequence, wherein the CRISPR complexcomprises the tracr mate sequence hybridized to the tracr sequence. Ingeneral, degree of complementarity is with reference to the optimalalignment of the tracr mate sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thetracr sequence or tracr mate sequence. In some embodiments, the degreeof complementarity between the tracr sequence and tracr mate sequencealong the length of the shorter of the two when optimally aligned isabout or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97.5%, 99%, or higher. Example illustrations of optimal alignmentbetween a tracr sequence and a tracr mate sequence are provided in FIGS.12B and 13B. In some embodiments, the tracr sequence is about or morethan about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, or more nucleotides in length. In some embodiments, thetracr sequence and tracr mate sequence are contained within a singletranscript, such that hybridization between the two produces atranscript having a secondary structure, such as a hairpin. Preferredloop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. In anembodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In some embodiments, the single transcript further includes atranscription termination sequence; preferably this is a polyT sequence,for example six T nucleotides. An example illustration of such a hairpinstructure is provided in the lower portion of FIG. 13B, where theportion of the sequence 5′ of the final “N” and upstream of the loopcorresponds to the tracr mate sequence, and the portion of the sequence3′ of the loop corresponds to the tracr sequence. Further non-limitingexamples of single polynucleotides comprising a guide sequence, a tracrmate sequence, and a tracr sequence are as follows (listed 5′ to 3′),where “N” represents a base of a guide sequence, the first block oflower case letters represent the tracr mate sequence, and the secondblock of lower case letters represent the tracr sequence, and the finalpoly-T sequence represents the transcription terminator: (1)NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (2)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT; (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT; (5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTT; and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT. In some embodiments, sequences (1) to (3) are used in combinationwith Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences(4) to (6) are used in combination with Cas9 from S. pyogenes. In someembodiments, the tracr sequence is a separate transcript from atranscript comprising the tracr mate sequence (such as illustrated inthe top portion of FIG. 13B).

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a CRISPR enzyme asa part of a CRISPR complex. A template polynucleotide may be of anysuitable length, such as about or more than about 10, 15, 20, 25, 50,75, 100, 150, 200, 500, 1000, or more nucleotides in length. In someembodiments, the template polynucleotide is complementary to a portionof a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or morenucleotides of a target sequences (e.g. about or more than about 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or morenucleotides). In some embodiments, when a template sequence and apolynucleotide comprising a target sequence are optimally aligned, thenearest nucleotide of the template polynucleotide is within about 1, 5,10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, ormore nucleotides from the target sequence.

In some embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GALA DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a CRISPR enzyme in combination with (and optionallycomplexed with) a guide sequence is delivered to a cell. Conventionalviral and non-viral based gene transfer methods can be used to introducenucleic acids in mammalian cells or target tissues. Such methods can beused to administer nucleic acids encoding components of a CRISPR systemto cells in culture, or in a host organism. Non-viral vector deliverysystems include DNA plasmids, RNA (e.g. a transcript of a vectordescribed herein), naked nucleic acid, and nucleic acid complexed with adelivery vehicle, such as a liposome. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt. Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression is preferred, adenoviralbased systems may be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and levels of expression havebeen obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors mayalso be used to transduce cells with target nucleic acids, e.g., in thein vitro production of nucleic acids and peptides, and for in vivo andex vivo gene therapy procedures (see, e.g., West et al., Virology160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, HumanGene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351(1994). Construction of recombinant AAV vectors are described in anumber of publications, including U.S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell.Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984);and Samulski et al., J. Virol. 63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7. HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, T1B55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-SF,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a CRISPR system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a CRISPR complex, is used to establisha new cell line comprising cells containing the modification but lackingany other exogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein. Transgenic animals are also provided, as are transgenic plants,especially crops and algae. The transgenic animal or plant may be usefulin applications outside of providing a disease model. These may includefood or feed production through expression of, for instance, higherprotein, carbohydrate, nutrient or vitamins levels than would normallybe seen in the wildtype. In this regard, transgenic plants, especiallypulses and tubers, and animals, especially mammals such as livestock(cows, sheep, goats and pigs), but also poultry and edible insects, arepreferred.

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence.

With recent advances in crop genomics, the ability to use CRISPR-Cassystems to perform efficient and cost effective gene editing andmanipulation will allow the rapid selection and comparison of single andand multiplexed genetic manipulations to transform such genomes forimproved production and enhanced traits. In this regard reference ismade to US patents and publications: U.S. Pat. No.6,603,061—Agrobacterium-Mediated Plant Transformation Method; U.S. Pat.No. 7,868,149—Plant Genome Sequences and Uses Thereof and US2009/0100536—Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics:advances andapplications” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96 are also hereinincorporated by reference in their entirety. In an advantageousembodiment of the invention, the CRISPR/Cas9 system is used to engineermicroalgae (Example 14). Accordingly, reference herein to animal cellsmay also apply, mutatis mutandis, to plant cells unless otherwiseapparent.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal or plant (includingmicro-algae), and modifying the cell or cells. Culturing may occur atany stage ex vivo. The cell or cells may even be re-introduced into thenon-human animal or plant (including micro-algae).

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system and instructions forusing the kit. In some embodiments, the vector system comprises (a) afirst regulatory element operably linked to a tracr mate sequence andone or more insertion sites for inserting a guide sequence upstream ofthe tracr mate sequence, wherein when expressed, the guide sequencedirects sequence-specific binding of a CRISPR complex to a targetsequence in a eukaryotic cell, wherein the CRISPR complex comprises aCRISPR enzyme complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the tracr mate sequence that ishybridized to the tracr sequence; and/or (b) a second regulatory elementoperably linked to an enzyme-coding sequence encoding said CRISPR enzymecomprising a nuclear localization sequence. Elements may provideindividually or in combinations, and may provided in any suitablecontainer, such as a vial, a bottle, or a tube. In some embodiments, thekit includes instructions in one or more languages, for example in morethan one language.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognised by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.

The target polynucleotide of a CRISPR complex may include a number ofdisease-associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed in USprovisional patent applications 61/736,527 and 61/748,427 having Broadreference BI-2011/008/WSGR Docket No. 44063-701.101and BI-2011/008/WSGRDocket No. 44063-701.102 respectively, both entitled SYSTEMS METHODS ANDCOMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec. 12, 2012 and Jan.2, 2013, respectively, the contents of all of which are hereinincorporated by reference in their entirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Examples of disease-associated genes and polynucleotides are availablefrom McKusick-Nathans Institute of Genetic Medicine, Johns HopkinsUniversity (Baltimore, Md.) and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, Md.), available onthe World Wide Web.

Examples of disease-associated genes and polynucleotides are listed inTables A and B. Disease specific information is available fromMcKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University(Baltimore, Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.), available on the WorldWide Web. Examples of signaling biochemical pathway-associated genes andpolynucleotides are listed in Table C.

Mutations in these genes and pathways can result in production ofimproper proteins or proteins in improper amounts which affect function.Further examples of genes, diseases and proteins are hereby incorporatedby reference from US Provisional applications 61/736,527 and 61/748,427.Such genes, proteins and pathways may be the target polynucleotide of aCRISPR complex.

TABLE A DISEASE/DISORDER GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2;ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; .AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor);FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor);TSG101; IGF; IGF Receptor; Igfl (4 variants); Igf2 (3 variants); Igf 1Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2,3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Abcr; Ccl2.; Cc2; cp(ceruloplasmin); Macular Timp3; cathepsinD; Degeneration Vldlrr; Ccr2Schizophrenia Neuregulin 1 (Nrg 1); Erb4 Disorders (receptor forNeuregulin); Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; 5-HTT (slc6a4); COMT;DRD (Drd1a); SLC6A3 DTNBP1; Dao (Dao1) Trinucleotide HTT (Huntington'sDx); Repeat SBMA/SMAX1/AR (Kennedy's Disorders Dx); FXN/X25 (Friedrich'sAtaxia); ATX3 (Machado- Joseph's Dx); ATXN1, and ATXN2 (spinocerebellarataxias); I)MPK (myotonic dystrophy); Atrophin-1 and Atn1 (DRPLA Dx);CBP (Creb-BP- global instability); VLDLR (Alzheimer's); Atxn7; Atxn10Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5 Secretase Related APH-1(alpha and beta): Disorders Presenilin (Psen1): nicastrin (Ncstn); PEN-2Others Nos1; Parp1; Nat 1; Nat2. Prion-related disorders Prp ALS SOD1;ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c) Drug addictionPrkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b;Grin2a; Drd3; Pdyn; Grial (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A;Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer’sDisease E1; CHIP; UGH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1;CR1; V1dlr; Ubal; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APPInflammation IL-10;1L-1 (IL-1a; IL-1b); 1L-13; 1L-17 (1L-17a (CTLA8);IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa;NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1Parkinson’s Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE B Blood and Anemia (CDAN1, CDAI, RPS19, DBA, PKLR, PKI,coagulation NT5C3, UMPHl, diseases PSN1, RHAG, RH50A., NRAMP2, SBTP, andALAS2, ANH1, ASB, disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FULJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, E1F2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell B-cell non-Hodgkin lymphoma (BCL7Adysregulation BCL7); Leukemia (TAL1, and TCL5, SCL, TAL2, FLT3, oncologyNBS1, NBS, ZNFN1A1, IK1, LYF1, diseases HOXD4, HOX4B; BCR, CML, PHL, andALL, ARNT, KRAS2, RASK2, disorders GMPS, AF10, ARHGEF12, LARG, KIAA0382,CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214,D9S46E, CAN, CAIN, RUNX1, CBFA2, AMLI, WHSC1L1, NSD3, FLT3, AF1Q, NPM1,NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1,P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11,PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1,NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN), InflammationAIDS (KIR3DL1, NKAT3, NKB1, AMB11, and KIR3DS1, IFNG, CXCL12, immuneSDF1); Autoimmune lymphoproliferative syndrome related (TNFRSF6, APT1,diseases FAS, CD95, ALPS1A); Combined and immunodeficiency, (IL2RG,disorders SCIDX1, SCIDX, 1MD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228),HIV susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5.CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2,TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX,AIID, XPID, PIDX, TNFRSF14B, TACI; Inflammation (IL-10, IL-1 (IL-1a,IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f),IL-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a,IL-12b), CTLA4, Cx3l1); Severe combined immunodeficiencies (SCIDs)(JAK3,JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R,CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4). Metabolic, Amyloid neuropathy(TTR, PALB); liver, Amyloidosis (APOA1, APP, AAA, kidney CVAP, AD1, GSN,FGA, LYZ, TTR, PALB); and protein Cirrhosis (KRT18, KRT8, diseasesCIRHIA, NAIC, TEX292, KIAA1988); and Cystic fibrosis (CFTR, ABCC7,disorders CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC,G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM);Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), Hepatic failure, earlyonset, and neurologic disorder (SCOD1, SCO1), Hepatic lipase deficiency(LIPC), Hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL, PDGRL,PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8,MCH5; Medullary cystic kidney disease (UMOD, HNFJ, FJHN, MCKD2,ADMCKD2); Phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); Polycystickidney and hepatic disease (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS,PRKCSH, G19P1, PCLD, SEC63). Muscular/ Becker muscular dystrophy (DMD,Skeletal BMD, MYF6), Duchenne Muscular diseases Dystrophy (DMD, BMD);and Emery-Dreifuss muscular dystrophy disorders (LMNA,LMN I, EMD2, FPLD,CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeralmuscular dystrophy (FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C,LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3,CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SOCA, ADL, DAG2, LGM)2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L TCAP, LGMD2G, CMD1N,TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J,POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1);Osteopetrosis (LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2,OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1); Muscular atrophy (VAPB, VAPC,ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D,HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1). Neurological ALS (SOD1, ALS2,STEX, FUS, TARDBP, and VEGF (VEGF-a, VEGF-b,VEGF-c); Alzheimer neuronaldisease (APP, AAA, CVAP, AD1, APOE, AD2, diseases PSEN2, AD4, STM2, andAPBB2, FE65L1, NOS3, PLAU, URK, ACE, disorders DCP1, ACE1, MPO, PACIP1PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1,MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3,NLGN4, KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5);Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP,JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT,TINUR, SNCAIP, TBP, SCA17, SNCA, NACP; PARK1, PARK4, DJ1, PARK7, LRRK2,PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUF-V2); Rett syndrome (MECP2, RTT, PPMX, MRX16,MRX79, CDKL5, , STK9, MECP2, RTT, PPMX, MRXI6 MRX79, x-Synuclein, DJ-1);Schizophrenia (Neuregulin1 (Nrg1) Erb4 (receptor for Neuregulin),Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophanhydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (S1c6a4), COW, DRD(Drd la), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders(APH-1 (alpha and beta), Presenitin (Pseni), nicastrin, (Ncstn), PEN-2,Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT(Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich'sAtaxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellarataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP- global instability), VLDLR (Alzheimer's), Atxn7, Atxn10).Occular Age-related macular degeneration diseases (Abcr, Ccl2, Cc2, cp(ceruloplasmin), and Timp3, cathepsinD, Vldlr, Ccr2); disorders Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYAI, GJA8, CX5O, CAEI , CJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGGI, CSD, BIGH3,CDG2, TACSTD2, TROP2, MIS1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLCIA, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1 NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP2O, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE C CELLULAR FUNCTION GENES PI3K/AKT PRKCE.; ITGAM; ITGA5; SignalingIRAK1; PRKAA2; EfF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKNJA; ITGB1; MAP2K2; JAM; AKTI; JAK2; PIK3RI; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2KI; NFKBI; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK.; CSNK1A1 ; BRAF; GSK3B; AKT3; FOXO1; SGK.; HSP90AA.1;RPS6KB1 ERK/MAPK PRKCE; ITGAM; ITGA5; Signaling HSPB1; IRAKI; PRKAA2;EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPKI; RAC2; PLK1; AKT2;PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3;MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1.; PPARG; PRKCD; PRKAA1;MAPK9; SRC; CDK2; PPP2CA; PIMI; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSRI;PXN; RAF1; FYN; DYRK1A; ITGB1 MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C;MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4;PRKCA; SRF; STAT1; SGK Glucocorticoid RAC1; TAF4B; EP300; SMAD2;Receptor TRAF6; PCAF; ELKI; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2;UBE2I; PIK3CA; CREBI; FOS; HSPA5; NFKB ; BCL2; MAP3K.14; STAT5B; PIK3C9;PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10; NRIPI; KRAS; MAPK13;RELA.; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2;SERPINE1; NCOA3; MAPK 14; TNF; RAF1; .IKBKG; MAP3K7; CREBBP; CDKN1A;MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1;NFKB1; TGFBRl; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1;IL6; HSP90AA1 Axonal PRKCE; ITGAM; ROCK1; Guidance ITGA5; CXCR4; ADAM12;Signaling IGFI; RACI; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO;ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2;CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABLI; MAPK3;ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN;ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1;PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCAEphrin PRKCE; ITGAM; ROCK1; Receptor ITGA5; CXCR4; IRAK1; SignalingPRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11;GNAS; PLK1; AKT2; DOKI; CDK8; CREB1.; PTK2; CFL1.; GNAQ; MAP3K14;CXCL12;MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1;MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2;PAK4; AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA;ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK ActinACTN4; PRKCE; ITGAM; ROCK1; Cytoskeleton ITGA5; IRAKI; Signaling PRKAA2;EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2;PIK3CA; CDK8; PTK2; CFL1 ; P1K3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R;MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRKIA; ITGB1; MAP2K2;PAK4; P1P5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC; ITGA2; TTK;CSNK1A1; CRKL; BRAF; VAV3; SGK Huntington’s PRKCE; IGF1; EP300; RCOR1;Disease PRKCZ; HDAC4; TGM2; Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2;SP1; CAPN2; PIK3CA; HDAC5; CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB;PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2;HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9;CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4;AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 Apoptosis PRKCE; ROCK1; BID; IRAK1;Signaling PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2;.IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2LI; CAPN1;MAPK3; CASPS; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF;RAFI; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKBI;PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3;BIRC3; PARP1 B Cell RACI; PTEN; LYN; Receptor ELK1; MAPK1; RAC2; PTPN11; Signaling AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14;PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA;PTPN6; MAPK9; EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7;MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6;BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte ACTN4; CD44;PRKCE; Extravasation ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A.;PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12;PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9;SRC; PIK3C2A.; BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2;CTNND1; PIK3R1 CTNNB1.; CLDN1 CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA;MMP1; MMP9 Integrin ACTN4; ITGAM; ROCK1; Signaling ITGA5; RAC1; PTEN;RAP1A; TLN1; ARHGEF7; MAPKI; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2;PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABLI; MAPK3; ITGA1; KRAS; RHOA; SRC;PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4;AKT1; PIK3RI; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF;GSK3B; AKT3 Acute IRAK1; SOD2; MYD88; Phase TRAF6; ELK1; Response MAPK1;PTPN11; Signaling AKT2; .IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB;MAPK8; RIPK1; MAPK3; 1L6ST; KRAS; MAPK13; IL6R; RELA; SOCSI; MAPK9; FTL;NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7;MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB;JUN; AKT3; IL1Rl.; IL6 PTEN ITGAM; ITGA5; RAC1; Signaling PTEN; PRKCZ;BCL2L11; MAPKI; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2;NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; .KRAS; ITGB7; ILK; PDGFRB;INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK;PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B;AKT3; FOXO1; CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN;BRCAT; GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB;PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1.; CHEK2; TNFRSF10B;TP73; RBI; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM213; APAFI; CTNNB1; SIRT1; CCND1 ; .PRKDC; ATM; SFN;CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3 Aryl HSPB1; EP300; FASN;Hydrocarbon TGM2; RXRA; MAPK1; NQO1; Receptor NCOR2; SP1; ARNT;Signaling CDKN1B; FOS; CHEK1; SMARCA4; NFKB2; MAPK8; ALDH1A1.; ATR;E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; SRC; CDK2; AHR;NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1; NFKB1 ; CCND1 ATM; ESR1;CDKN2A; MYC; JUN; ESR2; BAX; 1L6; CYP1B1; HSP90AA1 Xenobiotic PRKCE;EP300; PRKCZ; Metabolism RXRA; MAPK1; NQO1; Signaling NCOR2; PIK3CA;ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1;ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1;MAPK9; NOS2A; ABCB1;AHR; PPP2CA; FTL; NFE2L2; P1K3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP;MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKBI; KEAP1; PRKCA; EIF2AK3; IL6;CYP1B1; HSP90AAI SAPK/JNK PRKCE; IRAK1; PRKAA2; Signaling EIF2AK2; RAC1;ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8;PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS;PRKCD; PRKAA1; MARK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7;DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL;BRAF; SGK PPAr/RXR PRKAA2; EP300; INS; Signaling SMAD2; TRAF6; PPARA;FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2;MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A;NCOA3; MAPK14; INSR; RAFT; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2;CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1;ADIPOQ NF-KB IRAK1; EIF2AK2; EP300; Signaling INS; MYD88; PRKCZ; TRAF6;TBKI; AKT2; EGFR IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3;MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4; PDGFRB; TNF;INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA;NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1 Neuregulin ERBB4; PRKCE;ITGAM; Signaling ITGA5; PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2; EGFR;ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD; STAT5A;SRC; ITGB7; RAF1; ITGB1.; MAP2K2; ADAM 17; AKT1; PIK3R1; PDPKI ; MAP2K1;ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA;HSP90AA1; RPS6KB1 Wnt & Beta CD44; EP300; LRP6; catenin DVL3; CSNK1E;GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A;WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; LEF1; SOX9; TP53; MAP3K7;CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A;DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 Insulin PTEN; INS;EIF4E; Receptor PTPN1; PRKCZ; MAPK1; TSC1; Signaling PTPN11; AKT2; CBL;PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1;SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK;RPS6KB1 1L-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1; MAPKi; PTPN11;IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST; KRAS; MAPK13;IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB;MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1; CEBPB; JUN;IL1RI; SRF; IL6 Hepatic PRKCE; IRAK1; INS; MYD88; Cholestasis PRKCZ;TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10;RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7;IL8; CHUK; NR1H2; TJP2; NFKI31; ESR1; SREBF1; FGFR4; JUN; ILIRI; PRKCA.;1L6 IGF-1 IGF1; PRKCZ; ELK1; Signaling MAPK1; PTPN11; NEDD4; AKT2;PIK3CA; PRKC1; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1; MAPK3;IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR6I; AKT3; FOXO1; SRF; CTGF; RPS6KB1NRF2-mediated PRKCE; EP300; SOD2; Oxidative PRKCZ; MAPKI; SQSTM1; StressNQO1; PIK3CA; PRKCI; FOS; Response PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3;KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7;CREBBP; MAP2K2; AKT1; P1K3R1; MAP2K1; PPIB; JUN; KEAP1; GSK3B; ATF4;PRKCA; EIF2AK3; HSP90AA1 Hepatic EDN1; IGF1; KDR; FLT1; Fibrosis/ SMAD2;FGFR1; MET; PGF; Hepatic SMAD3; EGFR; FAS; CSF1; Stellate NFKB2; BCL2;MYH9; Cell IGHF1R; IL6R; RELA; TLR4; Activation PDGFRB; TNF; RELB; IL8;PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1;IL6; CTGF; MMP9 PPAR EP300; INS; TRAF6; PPARA; Signaling RXRA; MAPK1;IKBKG; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1; KRAS; PPARG;RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG; RELB;MAP3K7- CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1; NFKB1; JUN; IL1R1;HSP90AA1 Fc Epsilon PRKCE; RAC1; PRKCZ; RI Signaling LYN; MAPK1; RAC2;PTPN11; AKT2; PIK3CA; SYK; PRKCI; P1K3CB; PIK3C3; MAPK8; PRKD1; MAPK3;MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1;FYN; MAP2K2; AKT1; P1K3R1; PDPK1; MAP2K1; AKT3; VAV3; PRKCA G-ProteinPRKCE; .RAP1A ; RGS16; Coupled MAPK1; GNAS; AKT2; IKBKB; ReceptorPIK3CA; CREB1; GNAQ; Signaling NFKB2; CAMK2A; PIK3CB; PIK3C3: MAPK3;KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1;CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA InositolPRKCE; IRAK1; PRKAA2; Phosphate EIF2AK2; PTEN; GRK6; Metabolism MAPK1;PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1;MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1;PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF EIF2AK2; ELK1; ABL2.; SignalingMAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC;PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1;MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF ACTN4; ROCK1; KDR;Signaling FLT1.; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2;PIK3CB; P1K3C3;BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1;MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1.; SFN; VEGFA; AKT3; FOXO1; PRKCANatural PRKCE; RAC1; Killer Cell PRKCZ; MAPK1; Signaling RAC2; PTPN11;KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS;PRKCD; PTPN6; P1K3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1;MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: HDAC4; SMAD3; G1/S SUV39H1;HDAC5; CDKN1B; BTRC; Checkpoint ATR; ABL1; E2F1; Regulation HDAC2;HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1;E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6 T CellRAC1; ELK1; MAPK1; Receptor IKBKB; CBL; Signaling PIK3CA; FOS; NFKB2;PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK; LCK.; RAF1;IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN;VAV3 Death CRADD; HSPB1; BID; Receptor BIRC4; TBK1; IKBKB; SignalingFADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B;RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKBI; CASP2; BIRC2;CASP3; BIRC3 FGF RAC1; FGFR1; MET; Signaling MAPKAPK2; MAPK1; PTPN11;AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6;PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4;AKT3; PRKCA; HCF GM-CSF LYN; ELK1; MAPK1; Signaling PTPN11; AKT2;PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1;KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3;MAP2K1; CCND1; AKT3; STAT1 Amyotrophic BID; IGF1; RAC1; Lateral BIRC4;PGF; Sclerosis CAPNS1; CAPN2; Signaling PIK3CA; BCL2; PIK3CB; PIK30;BCL2L1.; CAPNI; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1 ;VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/ PTPNI; MAPK1; Stat PTPN11;AKT2; PIK3CA; Signaling STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1;STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1;STAT3; MAP2K1; FRAPI; AKT3; STAT1 Nicotinate PRKCE; IRAK1; and PRKAA2;EIF2AK2; Nicotinamide GRK6; MAPK1; Metabolism PLK1; AKT2; CDK8; MAPK8;MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2;MAP2K1;PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine CXCR4; ROCK2; MAPK1;Signaling PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12.; MAPK8; MAPK3; KRAS;MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1;JUN; CCL2; PRKCA 1L-2 ELK1; MAPK1; Signaling PTPN11; AKT2; PIK3CA; SYK;FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCSI; STAT5A; PIK3C2A;LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic PRKCE;IGF1; PRKCZ; Long PRDX6; LYN; MAPK1; Term GNAS; Depression PRKCI; GNAQ;PPP2R1A; IGFIR; PRKDI; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA;YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen TAF4B; EP300;CARM1; Receptor PCAF; MAPK1; NCOR2; Signaling SMARCA4; MAPK3; NRIP1;KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2;NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein TRAF6; SMURF1; BIRC4;Ubiquitination BRCA1; UCHLI; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5;USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1;VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS;NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7;JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/ PRKCE; EP300; RXR PRKCZ;RXRA; Activation GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD;RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCATGF- EP300; SMAD2; beta SMURF1; MAPK1; Signaling SMAD3; SMAD1; FOS;MAPK8; MAPK3; KRAS; MAPK9; RUNX2.; SERPINE1; RAF1; MAP3K7; CREBBP;MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll- IRAKI; EIF2AK2; likeMYD88; TRAF6; Receptor PPARA; ELK1; Signaling IKBKB; FOS; NFKB2;MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK;NFKB1; TLR2; JUN p38 HSPB1; IRAK1; TRAF6; MAPK Signaling MAPKAPK2; ELK1;FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK 14; TNF;MAP3K7; TGFBR1; MYC; ATF4; 1L1R1 ; SRF; STAT1 Neurotrophin/ NTRK2;MAPK1; PTPN11 ; TRK PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8;MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42;JUN; ATF4 FXR/ INS; PPARA; FASN; RXR RXRA; AKT2; Activation SDC1; MAPK8;APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1;FGFR4; AKT3; FOXO1 Synaptic PRKCE; RAP1A; Long EP300; PRKCZ; Term MAPK1;CREB1; Potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD;PPP1CC; RAFI; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium RAP1A; EP300;Signaling HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2;HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGFELK1; MAPK1; EGFR; Signaling PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3;PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1Hypoxia EDN1; PTEN; EP300; Signaling NQO1; UBE2I; in the CREB1; ARNT;Cardiovascular HIF1A; SLC2A4; System NOS3; TP53; LDHA; AKT1; ATM; VEGFA;JUN; ATF4; VHL; HSP90AA1 LPS/ IRAK1; MYD88; IL-1 TRAF6; PPARA; Mediated.RXRA; ABCA1; Inhibition MAPK8; ALDH1A1; of RXR Function GSTP1; MAPK9;ABCB1; TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 LXR/ FASN;RXRA; RXR NCOR2; ABCA1.; Activation NFKB2; IRF3; RELA; NOS2A; TLR4; TNF;RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 Amyloid PRKCE;CSNK1E; Processing MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3; MAPK13;MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP 1L-4 AKT2; PIK3CA;PIK3CB; Signaling PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A;JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: EP300; PCAF;G2/M BRCA1; GADD45A; DNA PLK1; BTRC; Damage CHEK1; ATR; CHEK2;Checkpoint YWHAZ; TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2ANitric KDR; FLT1; PGF; Oxide AKT2; PIK3CA; Signaling PIK3CB; PIK3C3; inCAV1; PRKCD; NOS3; PIK3C2A; the AKT1; PIK3R1; Cardiovascular SystemVEGFA.; AKT3; HSP90AA1 Purine NME2; SMARCA4; Metabolism MYH9; RRM2;ADAR.; EIF2AK4 PKM2; ENTPD1; RAD51; RRM2B; TJP2; .RAD51C; NT5E; POLD1;NME1 cAMP- RAP1A; MAPK1; GNAS; mediated CREB1; CAMK2A; MAPK3; SignalingSRC; RAH; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8;Dysfunction CASP8;MAPK10; MAPK9 CASP9; PARK7; PSEN1; PARK2; APP; CASP3Notch HES1; JAG1; NUMB; Signaling NOTCH4; ADAM17; NOTCH2; PSENI; NOTCH3;NOTCHI; DLL4 Endoplasmic HSPA5; MAPK8; Reticulum XBP1; TRAF2; StressATF6; CASP9; ATF4; Pathway ElF2AK3; CASP3 Pyrimidine NME2; AICDA; RRM2;Metabolism EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NMEI Parkinson’s UCHL1;MAPK8; MAPK13; Signaling MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac &GNAS; GNAQ; PPP2R1A; Beta GNB2L1; PPP2CA; Adrenergic PPP1CC; SignalingPPP2R5C Glycolysis/ HK2; GCK; GPI; Gluconeogenesis ALDH1A1; PKM2; LDHA;HK1 Interferon IRF1; SOCS1; JAK1; Signaling JAK2; IFITM1; STAT1; IFIT3Sonic ARRB2; SMO; GLI2; Hedgehog DYRK1A; GLI1; Signaling GSK3B; DYRK1BGlycero- PLD1; GRN; phospholipid. GPAM; YWHAZ; Metabolism SPHK1; SPHK2Phospholipid PRDX6; PLD1; GRN; Degradation YWHAZ; SPHK1; SPHK2Tryptophan SIAH2; PRMT5; NEDD4; Metabolism ALDH1A1; CYP1B1; SIAH1 LysineSUV39H1; EHMT2; Degradation NSD1; SETD7; PPP2R5C Nucleotide ERCC5;ERCC4; Excision XPA; XPC; ERCC1 Repair Pathway Starch and UCHL1; HK2;Sucrose GCK; GPI; HK1 Metabolism Aminosugars NQO1; HK2; Metabolism GCK;HK1 Arachidonic Acid PRDX6; Metabolism GRN; YWHAZ; CYP1B1 CircadianCSNK1E; CREB1; Rhythm ATF4; NR1D1 Signaling Coagulation BDKRB1; F2R;System SERPINE1; F3 Dopamine PPP2R1A; PPP2CA; Receptor PPP1CC; PPP2R5CSignaling Glutathione IDH2; GSTP1; Metabolism ANPEP; IDH1 GlycerolipidALDH1A1; GPAM; Metabolism SPHK1; SPHK2 Linoleic PRDX6; GRN; Acid YWHAZ;CYP1B1 Metabolism Methionine DNMT1; DNMT3B; Metabolism. AHCY; DNMT3APyruvate GLO1; ALDH1A1; Metabolism PKM2; LDHA Arginine ALDH1A1; andProline NOS3; NOS2A Metabolism Eicosanoid PRDX6; GRN; Signaling YWHAZFructose HK2; GCK; and Mannose HK1 Metabolism Galactose HK2; GCK;Metabolism HK1 Stilbene, PRDX6; PRDX1; Cournarine and TYR LigninBiosynthesis Antigen Presentation CALR; B2M Pathway Biosynthesis ofSteroids NQO1; DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate CycleIDH2; IDH1 Fatty Acid Metabolism ALDH1A1; CYP1B1 GlycerophospholipidPRDX6; CHKA Metabolism Histidine Metabolism PRMT5; ALDH1A1 InositolMetabolism ERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics byCytochrome p450 Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6;PRDX1 Metabolism Propanoate Metabolism ALDH1A1; LDHA Selenoamino AcidPRMT5; AHCY Metabolism Sphingolipid Metabolism SPHK1; SPHK2Aminophosphonate PRMT5 Metabolism Androgen and Estrogen PRMT5 MetabolismAscorbate and Aldarate ALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1Cysteine Metabolism LDHA Fatty Acid Biosynthesis FASN Glutamate ReceptorGNB2L1 Signaling NRF2-mediated PRDX1 Oxidative Stress Response PentosePhosphate GPI Pathway Pentose and Glucuronate UCHL1 interconversionsRetinol Metabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine MetabolismPRMT5, TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1Isoleucine Degradation Glycine, Serine and CHKA Threonine MetabolismLysine Degradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5;TRPC6; TRPC1; Cnrl; cnr2; Grk2; Trp1; Pomc; Cgrp; Crf; Pka; Era; Nr2b;TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial AIF; CytC; SMAC(Diablo); Function Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd);Neurology Noggin (Nog); WNT (Wnt2; Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6;Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Witt10b; Wnt16); beta-catenin;Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1;unc-86 (Pou4fl or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011—Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA*DNA hybrids. Melvor E I,Polak U. Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). TheCRISPR-Cas system may be harnessed to correct these defects of genomicinstability.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology:20; 2009).

In yet another aspect of the invention, the CRISPR-Cas system may be Clused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders. The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

In some embodiments, the condition may be neoplasia. In someembodiments, where the condition is neoplasia, the genes to be targetedare any of those listed in Table A (in this case PTEN asn so forth). Insome embodiments, the condition may be Age-related Macular Degeneration.In some embodiments, the condition may be a Schizophrenic Disorder. Insome embodiments, the condition may be a Trinucleotide Repeat Disorder.In some embodiments, the condition may be Fragile X Syndrome. In someembodiments, the condition may be a Secretase Related Disorder. In someembodiments, the condition may be a Prion—related disorder. In someembodiments, the condition may be ALS. In some embodiments, thecondition may be a drug addiction. In some embodiments, the conditionmay be Autism. In some embodiments, the condition may be Alzheimer'sDisease. In some embodiments, the condition may be inflammation. In someembodiments, the condition may be Parkinson's Disease.

Examples of proteins associated with Parkinson's disease include but arenot limited to α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1,Synphilin-1, and NURR1.

Examples of addiction-related proteins may include ABAT for example.

Examples of inflammation-related proteins may include the monocytechemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, the C-Cchemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgGreceptor IIB (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, orthe Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene, forexample.

Examples of cardiovascular diseases associated proteins may include IL1B(interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor proteinp53). PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin),IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-bindingcassette, sub-family G (WHITE), member 8), or CTSK (cathepsin K), forexample.

Examples of Alzheimer's disease associated proteins may include the verylow density lipoprotein receptor protein (VLDLR) encoded by the VLDLRgene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded bythe UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunitprotein (UBE1C) encoded by the UBA3 gene, for example.

Examples of proteins associated with Autism Spectrum Disorder mayinclude the benzodiazapine receptor (peripheral) associated protein 1(BZRAP1) encoded by the BZRAP1 gene, the AF4/FMR2 family member 2protein (AFF2) encoded by the AFF2 gene (also termed MFR2), the fragileX mental retardation autosomal homolog 1 protein (FXR1) encoded by theFXR1 gene, or the fragile X mental retardation autosomal homolog 2protein (FXR2) encoded by the FXR2 gene, for example.

Examples of proteins associated with Macular Degeneration may includethe ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, or the chemokine (C-C motif) Ligand 2 protein (CCL2)encoded by the CCL2 gene, for example.

Examples of proteins associated with Schizophrenia may include NRG1,ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, andcombinations thereof.

Examples of proteins involved in tumor suppression may include ATM(ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch2,Notch 3, or Notch 4, for example.

Examples of proteins associated with a secretase disorder may includePSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B),PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B(anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1),for example.

Examples of proteins associated with Amyotrophic Lateral Sclerosis mayinclude SOD1 (superoxide dismutase 1). ALS2 (amyotrophic lateralsclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein),VAGFA (vascular endothelial growth factor A), VAGFB (vascularendothelial growth factor B), and VAGFC (vascular endothelial growthfactor C), and any combination thereof.

Examples of proteins associated with prion diseases may include SOD1(superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS(fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascularendothelial growth factor A), VAGFB (vascular endothelial growth factorB), and VAGFC (vascular endothelial growth factor C), and anycombination thereof.

Examples of proteins related to neurodegenerative conditions in priondisorders may include A2M (Alpha-2-Macroglobulin), AATF (Apoptosisantagonizing transcription factor), ACPP (Acid phosphatase prostate),ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidasedomain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergicreceptor for Alpha-1D adrenoreceptor), for example.

Examples of proteins associated with Immunodeficiency may include A2M[alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase];ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2[ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3[ATP-binding cassette, sub-family A (ABC1), member 3]; for example.

Examples of proteins associated with Trinucleotide Repeat Disordersinclude AR (androgen receptor), FMR1 (fragile X mental retardation 1),HTT (huntingtin), or DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), for example.

Examples of proteins associated with Neurotransmission Disorders includeSST (somatostatin), NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A(adrenergic, alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-,receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine(serotonin) receptor 2C), for example.

Examples of neurodevelopmental-associated sequences include A2BP1[ataxin 2-binding protein 1], AADAT [aminoadipate aminotransferase],AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyrateaminotransferase], ABCA1 [ATP-binding cassette, sub-family A (ABC1),member 1], or ABCA13 [ATP-binding cassette, sub-family A (ABC1), member13], for example.

Further examples of preferred conditions treatable with the presentsystem include may be selected from: Aicardi-Goutieres Syndrome;Alexander Disease; Allan-Herndon-Dudley Syndrome; POLG-RelatedDisorders; Alpha-Mannosidosis (Type II and III); Alstr6m Syndrome;Angelman; Syndrome; Ataxia-Telangiectasia; NeuronalCeroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic Atrophy and(Infantile) Optic Atrophy Type 1; Retinoblastoma (bilateral); CanavanDisease; Cerebrooculofacioskeletal Syndrome 1 [COFS 1]; CerebrotendinousXanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders;Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial AlzheimerDisease; Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis; FukuyamaCongenital Muscular Dystrophy; Galactosialidosis; Gaucher Disease;Organic Acidemias; Hemophagocytic Lymphohistiocytosis;Hutchinson-Gilford Progeria Syndrome; Mucolipidosis 11; Infantile FreeSialic Acid Storage Disease; PLA2G6-Associated Neurodegeneration;Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa;Huntington Disease; Krabbe Disease (Infantile); MitochondrialDNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome;LIS1-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine Disease;MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders;LAMA2-Related Muscular Dystrophy; Arylsulfatase A Deficiency;Mucopolysaccharidosis Types I, II or III; Peroxisome BiogenesisDisorders, Zellweger Syndrome Spectrum; Neurodegeneration with BrainIron Accumulation Disorders; Acid Sphingomyelinase Deficiency;Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-RelatedDisorders; Urea Cycle Disorders; COL1A1/2-Related OsteogenesisImperfecta; Mitochondrial DNA Deletion Syndromes; PLP1-RelatedDisorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen StorageDisease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders;MECP2-Related Disorders; Rhizomelic Chondrodysplasia Punctata Type 1;Roberts Syndrome; Sandhoff Disease; Schindler Disease—Type 1; AdenosineDeaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal MuscularAtrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase ADeficiency; Thanatophoric Dysplasia Type 1; Collagen Type VI-RelatedDisorders; Usher Syndrome Type I; Congenital Muscular Dystrophy;Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; andXeroderma Pigmentosum.

Chronic administration of protein therapeutics may elicit unacceptableimmune responses to the specific protein. The immunogenicity of proteindrugs can be ascribed to a few immunodominant helper T lymphocyte (HTL)epitopes. Reducing the MHC binding affinity of these HTL epitopescontained within these proteins can generate drugs with lowerimmunogenicity (Tangri S, et al. (“Rationally engineered therapeuticproteins with reduced immunogenicity” J Immunol. 2005 Mar. 15;174(6):3187-96.) In the present invention, the immunogenicity of theCRISPR enzyme in particular may be reduced following the approach firstset out in Tangri et al with respect to erythropoietin and subsequentlydeveloped. Accordingly, directed evolution or rational design may beused to reduce the immunogenicity of the CRISPR enzyme (for instance aCas9) in the host species (human or other species).

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the Tables above and examples of genes currentlyassociated with those conditions are also provided there. However, thegenes exemplified are not exhaustive.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1 CRISPR Complex Activity in the Nucleus of a Eukaryotic Cell

An example type II CRISPR system is the type 11 CRISPR locus fromStreptococcus pyogenes SF370, which contains a cluster of four genesCas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements,tracrRNA and a characteristic array of repetitive sequences (directrepeats) interspaced by short stretches of non-repetitive sequences(spacers, about 30 bp each). In this system, targeted DNA double-strandbreak (DSB) is generated in four sequential steps (FIG. 2A). First, twonon-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed fromthe CRISPR locus. Second, tracrRNA hybridizes to the direct repeats ofpre-crRNA, which is then processed into mature crRNAs containingindividual spacer sequences. Third, the mature crRNA:tracrRNA complexdirects Cas9 to the DNA target consisting of the protospacer and thecorresponding PAM via heteroduplex formation between the spacer regionof the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage oftarget DNA upstream of PAM to create a DSB within the protospacer (FIG.2A). This example describes an example process for adapting thisRNA-programmable nuclease system to direct CRISPR complex activity inthe nuclei of eukaryotic cells.

To improve expression of CRISPR components in mammalian cells, two genesfrom the SF370 locus 1 of Streptococcus pyogenes (S. pyogenes) werecodon-optimized, Cas9 (SpCas9) and RNase III (SpRNase III). Tofacilitate nuclear localization, a nuclear localization signal (NLS) wasincluded at the amino (N)- or carboxyl (C)-termini of both SpCas9 andSpRNase III (FIG. 2B). To facilitate visualization of proteinexpression, a fluorescent protein marker was also included at the N- orC-termini of both proteins (FIG. 2B). A version of SpCas9 with an NLSattached to both N- and C-termini (2xNLS-SpCas9) was also generated.Constructs containing NLS-fused SpCas9 and SpRNase III were transfectedinto 293FT human embryonic kidney (HEK) cells, and the relativepositioning of the NLS to SpCas9 and SpRNase III was found to affecttheir nuclear localization efficiency. Whereas the C-terminal NLS wassufficient to target SpRNase III to the nucleus, attachment of a singlecopy of these particular NLS's to either the N- or C-terminus of SpCas9was unable to achieve adequate nuclear localization in this system. Inthis example, the C-terminal NLS was that of nucleoplasmin(KRPAATKKAGQAKKKK), and the C-terminal NLS was that of the SV40 largeT-antigen (PKKKRKV). Of the versions of SpCas9 tested, only 2xNLS-SpCas9exhibited nuclear localization (FIG. 2B).

The tracrRNA from the CRISPR locus of S. pyogenes SF370 has twotranscriptional start sites, giving rise to two transcripts of89-nucleotides (nt) and 171nt that are subsequently processed intoidentical 75nt mature tracrRNAs. The shorter 89nt tracrRNA was selectedfor expression in mammalian cells (expression constructs illustrated inFIG. 6, with functionality as determined by results of Surveryor assayshown in FIG. 6B). Transcription start sites are marked as +1, andtranscription terminator and the sequence probed by northern blot arealso indicated. Expression of processed tracrRNA was also confirmed byNorthern blot. FIG. 7C shows results of a Northern blot analysis oftotal RNA extracted from 293FT cells transfected with U6 expressionconstructs carrying long or short tracrRNA, as well as SpCas9 andDR-EMX1(1)-DR. Left and right panels are from 293FT cells transfectedwithout or with SpRNase III, respectively. U6 indicate loading controlblotted with a probe targeting human U6 snRNA. Transfection of the shorttracrRNA expression construct led to abundant levels of the processedform of tracrRNA (˜75 bp). Very low amounts of long tracrRNA aredetected on the Northern blot.

To promote precise transcriptional initiation, the RNA polymeraseIII-based U6 promoter was selected to drive the expression of tracrRNA(FIG. 2C). Similarly, a U6 promoter-based construct was developed toexpress a pre-crRNA array consisting of a single spacer flanked by twodirect repeats (DRs, also encompassed by the term “tracr-matesequences”; FIG. 2C). The initial spacer was designed to target a33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPRmotif (PAM) sequence satisfying the NGG recognition motif of Cas9) inthe human EALX1 locus (FIG. 2C), a key gene in the development of thecerebral cortex.

To test whether heterologous expression of the CRISPR system (SpCas9,SpRNase III, tracrRNA, and pre-crRNA) in mammalian cells can achievetargeted cleavage of mammalian chromosomes, HEK 293FT cells weretransfected with combinations of CRISPR components. Since DSBs inmammalian nuclei are partially repaired by the non-homologous endjoining (NHEJ) pathway, which leads to the formation of indels, theSurveyor assay was used to detect potential cleavage activity at thetarget EMX1 locus (see e.g. Guschin et al., 2010, Methods Mol Biol 649:247). Co-transfection of all four CRISPR components was able to induceup to 5.0% cleavage in the protospacer (see FIG. 2D). Co-transfection ofall CRISPR components minus SpRNase III also induced up to 4.7% indel inthe protospacer, suggesting that there may be endogenous mammalianRNases that are capable of assisting with crRNA maturation, such as forexample the related Dicer and Drosha enzymes. Removing any of theremaining three components abolished the genome cleavage activity of theCRISPR system (FIG. 2D). Sanger sequencing of amplicons containing thetarget locus verified the cleavage activity: in 43 sequenced clones, 5mutated alleles (11.6%) were found. Similar experiments using a varietyof guide sequences produced indel percentages as high as 29% (see FIGS.4-8, 10 and 11). These results define a three-component system forefficient CRISPR-mediated genome modification in mammalian cells.

To optimize the cleavage efficiency, Applicants also tested whetherdifferent isoforms of tracrRNA affected the cleavage efficiency andfound that, in this example system, only the short (89-bp) transcriptform was able to mediate cleavage of the human EM-AV genomic locus. FIG.9 provides an additional Northern blot analysis of crRNA processing inmammalian cells. FIG. 9A illustrates a schematic showing the expressionvector for a single spacer flanked by two direct repeats(DR-EMX1(1)-DR). The 30 bp spacer targeting the human EMX1 locusprotospacer 1 and the direct repeat sequences are shown in the sequencebeneath FIG. 9A. The line indicates the region whose reverse-complementsequence was used to generate Northern blot probes for EMX1(1) crRNAdetection. FIG. 9B shows a Northern blot analysis of total RNA extractedfrom 293FT cells transfected with U6 expression constructs carryingDR-EMX1(1)-DR. Left and right panels are from 293FT cells transfectedwithout or with SpRNase III respectively. DR-EMX1(1)-DR was processedinto mature crRNAs only in the presence of SpCas9 and short tracrRNA andwas not dependent on the presence of SpRNase III. The mature crRNAdetected from transfected 293FT total RNA is ˜33 bp and is shorter thanthe 39-42 bp mature crRNA from S. pyogenes. These results demonstratethat a CRISPR system can be transplanted into eukaryotic cells andreprogrammed to facilitate cleavage of endogenous mammalian targetpolynucleotides.

FIG. 2 illustrates the bacterial CRISPR system described in thisexample. FIG. 2A illustrates a schematic showing the CRISPR locus 1 fromStreptococcus pyogenes SF370 and a proposed mechanism of CRISPR-mediatedDNA cleavage by this system. Mature crRNA processed from the directrepeat-spacer array directs Cas9 to genomic targets consisting ofcomplimentary protospacers and a protospacer-adjacent motif (PAM). Upontarget-spacer base pairing, Cas9 mediates a double-strand break in thetarget DNA. FIG. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9)and RNase III (SpRNase III) with nuclear localization signals (NLSs) toenable import into the mammalian nucleus. FIG. 2C illustrates mammalianexpression of SpCas9 and SpRNase III driven by the constitutive EF1apromoter and tracrRNA and pre-crRNA array (DR-Spacer-DR) driven by theRNA Pol3 promoter U6 to promote precise transcription initiation andtermination. A protospacer from the human EMX1 locus with a satisfactoryPAM sequence is used as the spacer in the pre-crRNA array. FIG. 2Dillustrates surveyor nuclease assay for SpCas9-mediated minor insertionsand deletions. SpCas9 was expressed with and without SpRNase III,tracrRNA, and a pre-crRNA array carrying the EMX1-target spacer. FIG. 2Eillustrates a schematic representation of base pairing between targetlocus and EMX1-targeting crRNA, as well as an example chromatogramshowing a micro deletion adjacent to the SpCas9 cleavage site. FIG. 2Fillustrates mutated alleles identified from sequencing analysis of 43clonal amplicons showing a variety of micro insertions and deletions.Dashes indicate deleted bases, and non-aligned or mismatched basesindicate insertions or mutations. Scale bar=10 μm.

To further simplify the three-component system, a chimericcrRNA-tracrRNA hybrid design was adapted, where a mature crRNA(comprising a guide sequence) is fused to a partial tracrRNA via astem-loop to mimic the natural crRNA:tracrRNA duplex (FIG. 3A).

Guide sequences can be inserted between BbsI sites using annealedoligonucleotides. Protospacers on the sense and anti-sense strands areindicated above and below the DNA sequences, respectively. Amodification rate of 6.3% and 0.75% was achieved for the human PVALB andmouse Th loci respectively, demonstrating the broad applicability of theCRISPR system in modifying different loci across multiple organismsWhile cleavage was only detected with one out of three spacers for eachlocus using the chimeric constructs, all target sequences were cleavedwith efficiency of indel production reaching 27% when using theco-expressed pre-crRNA arrangement (FIGS. 4 and 5).

FIG. 5 provides a further illustration that SpCas9 can be reprogrammedto target multiple genomic loci in mammalian cells. FIG. 5A provides aschematic of the human EMX1 locus showing the location of fiveprotospacers, indicated by the underlined sequences. FIG. 5B provides aschematic of the pre-crRNA/trcrRNA complex showing hybridization betweenthe direct repeat region of the pre-crRNA and tracrRNA (top), and aschematic of a chimeric RNA design comprising a 20 bp guide sequence,and tracr mate and tracr sequences consisting of partial direct repeatand tracrRNA sequences hybridized in a hairpin structure (bottom).Results of a Surveyor assay comparing the efficacy of Cas9-mediatedcleavage at five protospacers in the human EMX1 locus is illustrated inFIG. 5C. Each protospacer is targeted using either processedpre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).

Since the secondary structure of RNA can be crucial for intermolecularinteractions, a structure prediction algorithm based on minimum freeenergy and Boltzmann-weighted structure ensemble was used to compare theputative secondary structure of all guide sequences used in our genometargeting experiment (FIG. 3B) (see e.g. Gruber et al., 2008, NucleicAcids Research, 36: W70). Analysis revealed that in most cases, theeffective guide sequences in the chimeric crRNA context weresubstantially free of secondary structure motifs, whereas theineffective guide sequences were more likely to form internal secondarystructures that could prevent base pairing with the target protospacerDNA. It is thus possible that variability in the spacer secondarystructure might impact the efficiency of CRISPR-mediated interferencewhen using a chimeric crRNA.

FIG. 3 illustrates example expression vectors. FIG. 3A provides aschematic of a bi-cistronic vector for driving the expression of asynthetic crRNA-tracrRNA chimera (chimeric RNA) as well as SpCas9. Thechimeric guide RNA contains a 20-bp guide sequence corresponding to theprotospacer in the genomic target site. FIG. 3B provides a schematicshowing guide sequences targeting the human EMX1, PVALB, and mouse Thloci, as well as their predicted secondary structures. The modificationefficiency at each target site is indicated below the RNA secondarystructure drawing (EMX1, n=216 amplicon sequencing reads; PVALB, n=224reads; Th, n=265 reads). The folding algorithm produced an output witheach base colored according to its probability of assuming the predictedsecondary structure, as indicated by a rainbow scale that is reproducedin FIG. 3B in gray scale. Further vector designs for SpCas9 are shown inFIG. 3A, including single expression vectors incorporating a U6 promoterlinked to an insertion site for a guide oligo, and a Cbh promoter linkedto SpCas9 coding sequence.

To test whether spacers containing secondary structures are able tofunction in prokaryotic cells where CRISPRs naturally operate,transformation interference of protospacer-bearing plasmids were testedin an E. coli strain heterologously expressing the S. pyogenes SF370CRISPR locus 1 (FIG. 3C). The CRISPR locus was cloned into a low-copy E.coli expression vector and the crRNA array was replaced with a singlespacer flanked by a pair of DRs (pCRISPR). E. coli strains harboringdifferent pCRISPR plasmids were transformed with challenge plasmidscontaining the corresponding protospacer and PAM sequences (FIG. 3C). Inthe bacterial assay, all spacers facilitated efficient CRISPRinterference (FIG. 3C). These results suggest that there may beadditional factors affecting the efficiency of CRISPR activity inmammalian cells.

To investigate the specificity of CRISPR-mediated cleavage, the effectof single-nucleotide mutations in the guide sequence on protospacercleavage in the mammalian genome was analyzed using a series ofEMX1-targeting chimeric crRNAs with single point mutations (FIG. 4A).FIG. 4B illustrates results of a Surveyor nuclease assay comparing thecleavage efficiency of Cas9 when paired with different mutant chimericRNAs. Single-base mismatch up to 12-bp 5′ of the PAM substantiallyabrogated genomic cleavage by SpCas9, whereas spacers with mutations atfarther upstream positions retained activity against the originalprotospacer target (FIG. 4B). In addition to the PAM, SpCas9 hassingle-base specificity within the last 12-bp of the spacer.Furthermore, CRISPR is able to mediate genomic cleavage as efficientlyas a pair of TALE nucleases (TALEN) targeting the same EMX1 protospacer.FIG. 4C provides a schematic showing the design of TALENs targeting EEMX1, and FIG. 4D shows a Surveyor gel comparing the efficiency of TALENand Cas9 (n=3).

Having established a set of components for achieving CRISPR-mediatedgene editing in mammalian cells through the error-prone NHEJ mechanism,the ability of CRISPR to stimulate homologous recombination (HR), a highfidelity gene repair pathway for making precise edits in the genome, wastested. The wild type SpCas9 is able to mediate site-specific DSBs,which can be repaired through both NHEJ and HR. In addition, anaspartate-to-alanine substitution (D10A) in the RuvC I catalytic domainof SpCas9 was engineered to convert the nuclease into a nickase(SpCas9n; illustrated in FIG. 5A) (see e.g. Sapranausaks et al., 2011,Cucleic Acis Research, 39: 9275; Gasiunas et al., 2012, Proc. Natl.Acad. Sci. USA, 109:E2579), such that nicked genomic DNA undergoes thehigh-fidelity homology-directed repair (HDR). Surveyor assay confirmedthat SpCas9n does not generate indels at the EMX1 protospacer target. Asillustrated in FIG. 5B, co-expression of EMX1-targeting chimeric crRNAwith SpCas9 produced indels in the target site, whereas co-expressionwith SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons didnot detect any indels induced by SpCas9n. The same locus was selected totest CRISPR-mediated HR by co-transfecting HEK 293FT cells with thechimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HRtemplate to introduce a pair of restriction sites (HindIII and NheI)near the protospacer. FIG. 5C provides a schematic illustration of theHR strategy, with relative locations of recombination points and primerannealing sequences (arrows). SpCas9 and SpCas9n indeed catalyzedintegration of the HR template into the EMX1 locus. PCR amplification ofthe target region followed by restriction digest with HindIII revealedcleavage products corresponding to expected fragment sizes (arrows inrestriction fragment length polymorphism gel analysis shown in FIG. 5D),with SpCas9 and SpCas9n mediating similar levels of HR efficiencies.Applicants further verified HR using Sanger sequencing of genomicamplicons (FIG. 5E). These results demonstrate the utility of CRISPR forfacilitating targeted gene insertion in the mammalian genome. Given the14-bp (12-bp from the spacer and 2-bp from the PAM) target specificityof the wild type SpCas9, the availability of a nickase can significantlyreduce the likelihood of off-target modifications, since single strandbreaks are not substrates for the error-prone NHEJ pathway.

Expression constructs mimicking the natural architecture of CRISPR lociwith arrayed spacers (FIG. 2A) were constructed to test the possibilityof multiplexed sequence targeting. Using a single CRISPR array encodinga pair of EMX1- and PVALB-targeting spacers, efficient cleavage at bothloci was detected (FIG. 4F, showing both a schematic design of the crRNAarray and a Surveyor blot showing efficient mediation of cleavage).Targeted deletion of larger genomic regions through concurrent DSBsusing spacers against two targets within EMX1 spaced by 119 bp was alsotested, and a 1.6% deletion efficacy (3 out of 182 amplicons; FIG. 5G)was detected. This demonstrates that the CRISPR system can mediatemultiplexed editing within a single genome.

Example 2 CRISPR System Modifications and Alternatives

The ability to use RNA to program sequence-specific DNA cleavage definesa new class of genome engineering tools for a variety of research andindustrial applications. Several aspects of the CRISPR system can befurther improved to increase the efficiency and versatility of CRISPRtargeting. Optimal Cas9 activity may depend on the availability of freeMg²⁺ at levels higher than that present in the mammalian nucleus (seee.g. Jinek et al., 2012. Science, 337:816), and the preference for anNGG motif immediately downstream of the protospacer restricts theability to target on average every 12-bp in the human genome. Some ofthese constraints can be overcome by exploring the diversity of CRISPRloci across the microbial metagenome (see e.g. Makarova et al., 2011,Nat Rev Microbiol, 9:467). Other CRISPR loci may be transplanted intothe mammalian cellular milieu by a process similar to that described inExample 1. The modification efficiency at each target site is indicatedbelow the RNA secondary structures. The algorithm generating thestructures colors each base according to its probability of assuming thepredicted secondary structure. RNA guide spacers 1 and 2 induced 14% and6.4%, respectively. Statistical analysis of cleavage activity acrossbiological replica at these two protospacer sites is also provided inFIG. 7.

Example 3 Sample Target Sequence Selection Algorithm

A software program is designed to identify candidate CRISPR targetsequences on both strands of an input DNA sequence based on desiredguide sequence length and a CRISPR motif sequence (PAM) for a specifiedCRISPR enzyme. For example, target sites for Cas9 from S. pyogenes, withPAM sequences NGG, may be identified by searching for 5′-N_(x)-NGG-3′both on the input sequence and on the reverse-complement of the input.Likewise, target sites for Cas9 of S. thermophilus CRISPR1, with PAMsequence NNAGAAW, may be identified by searching for 5′-N_(x)-NNAGAAW-3′both on the input sequence and on the reverse-complement of the input.Likewise, target sites for Cas9 of S. thermophilus CRISPR3, with PAMsequence NGGNG, may be identified by searching for 5′-N,-NGGNG-3′ bothon the input sequence and on the reverse-complement of the input. Thevalue “x” in N_(x) may be fixed by the program or specified by the user,such as 20.

Since multiple occurrences in the genome of the DNA target site may leadto nonspecific genome editing, after identifying all potential sites,the program filters out sequences based on the number of times theyappear in the relevant reference genome. For those CRISPR enzymes forwhich sequence specificity is determined by a ‘seed’ sequence, such asthe 11-12 bp 5′ from the PAM sequence, including the PAM sequenceitself, the filtering step may be based on the seed sequence. Thus, toavoid editing at additional genomic loci, results are filtered based onthe number of occurrences of the seed:PAM sequence in the relevantgenome. The user may be allowed to choose the length of the seedsequence. The user may also be allowed to specify the number ofoccurrences of the seed:PAM sequence in a genome for purposes of passingthe filter. The default is to screen for unique sequences. Filtrationlevel is altered by changing both the length of the seed sequence andthe number of occurrences of the sequence in the genome. The program mayin addition or alternatively provide the sequence of a guide sequencecomplementary to the reported target sequence(s) by providing thereverse complement of the identified target sequence(s).

Further details of methods and algorithms to optimize sequence selectioncan be found found in U.S. application Ser. No. TBA (Broad ReferenceBI-2012/084 44790.11.2022); incorporated herein by reference.

Example 4 Evaluation of Multiple Chimeric crRNA-tracrRNA Hybrids

This example describes results obtained for chimeric RNAs (chiRNAs;comprising a guide sequence, a tracr mate sequence, and a tracr sequencein a single transcript) having tracr sequences that incorporatedifferent lengths of wild-type tracrRNA sequence. FIG. 18 a illustratesa schematic of a bicistronic expression vector for chimeric RNA andCas9. Cas9 is driven by the CBh promoter and the chimeric RNA is drivenby a U6 promoter. The chimeric guide RNA consists of a 20 bp guidesequence (Ns) joined to the tracr sequence (running from the first “U”of the lower strand to the end of the transcript), which is truncated atvarious positions as indicated. The guide and tracr sequences areseparated by the tracr-mate sequence GUUUUAGAGCUA followed by the loopsequence GAAA. Results of SURVEYOR assays for Cas9-mediated indels atthe human EMX1 and PVALB loci are illustrated in FIGS. 18 b and 18 c,respectively. Arrows indicate the expected SURVEYOR fragments. ChiRNAsare indicated by their “+n” designation, and crRNA refers to a hybridRNA where guide and tracr sequences are expressed as separatetranscripts. Quantification of these results, performed in triplicate,are illustrated by histogram in FIGS. 11 a and 11 b, corresponding toFIGS. 10 b and 10 c, respectively (“N.D.” indicates no indels detected).Protospacer IDs and their corresponding genomic target, protospacersequence, PAM sequence, and strand location are provided in Table D.Guide sequences were designed to be complementary to the entireprotospacer sequence in the case of separate transcripts in the hybridsystem, or only to the underlined portion in the case of chimeric RNAs.

TABLE D protospacer genomic ID target protospacer sequence (5′ to 3′)PAM Strand 1 EMX1 GGACATCGATGTCACCTCCAATGACTAG TGG + GG 2 EMX1CATTGGAGGTGACATCGATGTCCTCCCC TGG − AT 3 EMX1 GGAAGGGCCTGAGTCCGAGCAGAAGAAGGG + GAA 4 PVALB GGTGGCGAGAGGGGCCGAGATTGGGTGT AGG + TC 5 PVALBATGCAGGAGGGTGGCGAGAGGGGCCGA TGG + GAT

Cell Culture and Transfection

Human embryonic kidney (HEK) cell line 293FT (Life Technologies) wasmaintained in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (LifeTechnologies), 100U/mL penicillin, and 100 μg/mL streptomycin at 37° C.with 5% CO₂ incubation. 293FT cells were seeded onto 24-well plates(Corning) 24 hours prior to transfection at a density of 150,000 cellsper well. Cells were transfected using Lipofectamine 2000 (LifeTechnologies) following the manufacturer's recommended protocol. Foreach well of a 24-well plate, a total of 500 ng plasmid was used.

SURVEYOR Assay for Genome Modification

293FT cells were transfected with plasmid DNA as described above. Cellswere incubated at 37° C. for 72 hours post-transfection prior to genomicDNA extraction. Genomic DNA was extracted using the QuickExtract DNAExtraction Solution (Epicentre) following the manufacturer's protocol.Briefly, pelleted cells were resuspended in QuickExtract solution andincubated at 65° C. for 15 minutes and 98° C. for 10 minutes. Thegenomic region flanking the CRISPR target site for each gene was PCRamplified (primers listed in Table E), and products were purified usingQiaQuick Spin Column (Qiagen) following the manufacturer's protocol. 400ng total of the purified PCR products were mixed with 2 μl 10×Taq DNAPolymerase PCR buffer (Enzymatics) and ultrapure water to a final volumeof 20 μl, and subjected to a re-annealing process to enable heteroduplexformation: 95° C. for 10 min, 95° C. to 85° C. ramping at −2° C./s. 85°C. to 25° C. at −0.25° C./s, and 25° C. hold for 1 minute. Afterre-annealing, products were treated with SURVEYOR nuclease and SURVEYORenhancer S (Transgenomics) following the manufacturer's recommendedprotocol, and analyzed on 4-20% Novex TBE poly-acrylamide gels (LifeTechnologies). Gels were stained with SYBR Gold DNA stain (LifeTechnologies) for 30 minutes and imaged with a Gel Doc gel imagingsystem (Bio-rad). Quantification was based on relative band intensities.

TABLE E genomic primer name target primer sequence (5′ to 3′) Sp-EMX1-FEMX1 AAAACCACCCTTCTCTCTGGC Sp-EMX1-R EMX1 GGAGATTGGAGACACGGAGAGSp-PVALB-F PVALB CTGGAAAGCCAATGCCTGAC Sp-PVALB-R PVALBGGCAGCAAACTCCTTGTCCT

Computational Identification of Unique CRISPR Target Sites

To identify unique target sites for the S. pyogenes SF370 Cas9 (SpCas9)enzyme in the human, mouse, rat, zebrafish, fruit fly, and C. elegansgenome, we developed a software package to scan both strands of a DNAsequence and identify all possible SpCas9 target sites. For thisexample, each SpCas9 target site was operationally defined as a 20 bpsequence followed by an NGG protospacer adjacent motif (PAM) sequence,and we identified all sequences satisfying this 5′-N₂₀-NGG-3′ definitionon all chromosomes. To prevent non-specific genome editing, afteridentifying all potential sites, all target sites were filtered based onthe number of times they appear in the relevant reference genome. Totake advantage of sequence specificity of Cas9 activity conferred by a‘seed’ sequence, which can be, for example, approximately 11-12 bpsequence 5′ from the PAM sequence, 5′-NNNNNNNNNN-NGG-3′ sequences wereselected to be unique in the relevant genome. All genomic sequences weredownloaded from the UCSC Genome Browser (Human genome hg19, Mouse genomemm9, Rat genome rn5, Zebrafish genome danRer7, D. melanogaster genomedm4 and C. elegans genome ce10). The full search results are availableto browse using UCSC Genome Browser information. An examplevisualization of some target sites in the human genome is provided inFIG. 22.

Initially, three sites within the EMX1 locus in human HEK 293FT cellswere targeted. Genome modification efficiency of each chiRNA wasassessed using the SURVEYOR nuclease assay, which detects mutationsresulting from DNA double-strand breaks (DSBs) and their subsequentrepair by the non-homologous end joining (NHEJ) DNA damage repairpathway. Constructs designated chiRNA(+n) indicate that up to the +nnucleotide of wild-type tracrRNA is included in the chimeric RNAconstruct, with values of 48, 54, 67, and 85 used for n. Chimeric RNAscontaining longer fragments of wild-type tracrRNA (chiRNA(+67) andchiRNA(+85)) mediated DNA cleavage at all three EMX1 target sites, withchiRNA(+85) in particular demonstrating significantly higher levels ofDNA cleavage than the corresponding crRNA/tracrRNA hybrids thatexpressed guide and tracr sequences in separate transcripts (FIGS. 10 band 10 a). Two sites in the PVALB locus that yielded no detectablecleavage using the hybrid system (guide sequence and tracr sequenceexpressed as separate transcripts) were also targeted using chiRNAs.chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage atthe two PVALB protospacers (FIGS. 10 c and 10 b).

For all five targets in the EMX1 and PVALB loci, a consistent increasein genome modification efficiency with increasing tracr sequence lengthwas observed. Without wishing to be bound by any theory, the secondarystructure formed by the 3′ end of the tracrRNA may play a role inenhancing the rate of CRISPR complex formation. An illustration ofpredicted secondary structures for each of the chimeric RNAs used inthis example is provided in FIG. 21. The secondary structure waspredicted using RNAfold(http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) using minimum freeenergy and partition function algorithm. Pseudocolor for each based(reproduced in grayscale) indicates the probability of pairing. BecausechiRNAs with longer tracr sequences were able to cleave targets thatwere not cleaved by native CRISPR crRNA/tracrRNA hybrids, it is possiblethat chimeric RNA may be loaded onto Cas9 more efficiently than itsnative hybrid counterpart. To facilitate the application of Cas9 forsite-specific genome editing in eukaryotic cells and organisms, allpredicted unique target sites for the S. pyogenes Cas9 werecomputationally identified in the human, mouse, rat, zebra fish, C.elegans, and D. melanogaster genomes. Chimeric RNAs can be designed forCas9 enzymes from other microbes to expand the target space of CRISPRRNA-programmable nucleases.

FIGS. 11 and 21 illustrate exemplary bicistronic expression vectors forexpression of chimeric RNA including up to the +85 nucleotide ofwild-type tracr RNA sequence, and SpCas9 with nuclear localizationsequences. SpCas9 is expressed from a CBh promoter and terminated withthe bGH polyA signal (bGH pA). The expanded sequence illustratedimmediately below the schematic corresponds to the region surroundingthe guide sequence insertion site, and includes, from 5′ to3′,3′-portion of the U6 promoter (first shaded region), BbsI cleavagesites (arrows), partial direct repeat (tracr mate sequence GTTTTAGAGCTA,underlined), loop sequence GAAA, and +85 tracr sequence (underlinedsequence following loop sequence). An exemplary guide sequence insert isillustrated below the guide sequence insertion site, with nucleotides ofthe guide sequence for a selected target represented by an “N”.

Sequences described in the above examples are as follows (polynucleotidesequences are 5′ to 3′):

U6-short tracrRNA (Streptococcus pyogenes SF370):

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTTTTT (bold = tracrRNA sequence;underline = terminator sequence)

U6-long tracrRNA (Streptococcus pyogenes SF370):

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGTAGTATTAAGTATTGTTTTATGGCTGATAAATTTCTTTGAATTTCTCCTTGATTATTTGTTATAAAAGTTATAAAATAATCTTGTTGGAACCATTCAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

U6-DR-BbsI backbone-DR (Streptococcus pyogenes SF370):

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACGGGTCTTCGAGAAGACGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC

U6-chimeric RNA-BbsI backbone (Streptococcus pyogenes SF370)

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG GCTAGTCCG

NLS-SpCas9-EGFP:

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKLEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

SpCas9-EGFP-NLS:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKRPAATKKAGQAKKKK

NLS-SpCas9-EGFP-NLS:

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYTDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEEVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDAAAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKR PAATKKAGQAKKKK

NLS-SpCas9-NLS:

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKMKNYWQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKLRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK

NLS-mCherry-SpRNase3:

MFLFLSLTSFLSSSRTLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGSKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEV

SpRNase3-mCherry-NLS:

MKQLEELLSTSFDIQFNDLTLLETAFTHTSYANEHRLLNVSHNERLEFLGDAVLQLIISEYLFAKYPKKTEGDMSKLRSMIVREESLAGFSRFCSFDAYIKLGKGEEKSGGRRRDTILGDLFEAFLGALLLDKGIDAVRRFLKQVMIPQVEKGNFERVKDYKTCLQEFLQTKGDVAIDYQVISEKGPAHAKQFEVSIVVNGAVLSKGLGKSKKLAEQDAAKNALAQLSEVGSVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKKRPAATKKAGQAKKKK

NLS-SpCas9n-NLS (the D10A nickase mutation is lowercase):

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLaIGTNSVGWAVITDEYKWSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK

hEMX1-HR Template-HindII-NheI:

GAATGCTGCCCTCAGACCCGCTTCCTCCCTGTCCTTGTCTGTCCAAGGAGAATGAGGTCTCACTGGTGGATTTCGGACTACCCTGAGGAGCTGGCACCTGAGGGACAAGGCCCCCCACCTGCCCAGCTCCAGCCTCTGATGAGGGGTGGGAGAGAGCTACATGAGGTTGCTAAGAAAGCCTCCCCTGAAGGAGACCACACAGTGTGTGAGGTTGGAGTCTCTAGCAGCGGGTTCTGTGCCCCCAGGGATAGTCTGGCTGTCCAGGCACTGCTCTTGATAAAACACCACCTCCTAGTTATGAAACCATGCCCATTCTGCCTCTCTGTATGGAAAAGAGCATGGGGCTGGCCCGTGGGGTGGTGTCCACTTTAGGCCCTGTGGGAGATCATGGGAACCCACGCAGTGGGTCATAGGCTCTCTCATTTACTACTCACATCCACTCTGTGAAGAAGCGATTATGATCTCTCCTCTAGAAACTCGTAGAGTCCCATGTCTGCCGGCTTCCAGAGCCTGCACTCCTCCACCTTGGCTTGGCTTTGCTGGGGCTAGAGGAGCTAGGATGCACAGCAGCTCTGTGACCCTTTGTTTGAGAGGAACAGGAAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTTCCTGCCCTGCCATCCCCTTCTGTGAATGTTAGACCCATGGGAGCAGCTGGTCAGAGGGGACCCCGGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTCTTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACaagcttgctagcGGTGGGCAACCACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAGGGACAAGCAATGGGCTGGCTGAGGCCTGGGACCACTTGGCCTTCTCCTCGGAGAGCCTGCCTGCCTGGGCGGGCCCGCCCGCCACCGCAGCCTCCCAGCTGCTCTCCGTGTCTCCAATCTCCCTTTTGTTTTGATGCATTTCTGTTTTAATTTATTTTCCAGGCACCACTGTAGTTTAGTGATCCCCAGTGTCCCCCTTCCCTATGGGAATAATAAAAGTCTCTCTCTTAATGACACGGGCATCCAGCTCCAGCCCCAGAGCCTGGGGTGGTAGATTCCGGCTCTGAGGGCCAGTGGGGGCTGGTAGAGCAAACGCGTTCAGGGCCTGGGAGCCTGGGGTGGGGTACTGGTGGAGGGGGTCAAGGGTAATTCATTAACTCCTCTCTTTTGTTGGGGGACCCTGGTCTCTACCTCCAGCTCCACAGCAGGAGAAACAGGCTAGACATAGGGAAGGGCCATCCTGTATCTTGAGGGAGGACAGGCCCAGGTCTTTCTTAACGTATTGAGAGGTGGGAATCAGGCCCAGGTAGTTCAATGGGAGAGGGAGAGTGCTTCCCTCTGCCTAGAGACTCTGGTGGCTTCTCCAGTTGAGGAGAAACCAGAGGAAAGGGGAGGATTGGGGTCTGGGGGAGGGAACACCATTCACAAAGGCTGACGGTTCCAGTCCGAAGTCGTGGGCCCACCAGGATGCTCACCTGTCCTTGGAGAACCGCTGGGCAGGTTGAGACTGCAGAGACAGGGCTTAAGGCTGAGCCTGCAACCAGTCCCCAGTGACTCAGGGCCTCCTCAGCCCAAGAAAGAGCAACGTGCCAGGGCCCGCTGAGCTCTT GTGTTCACCTG

NLS-StCsn1-NLS:

MKRPAATKKAGQAKKKKSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFKRPAATKKAGQAK KKK

U6-St_tracrRNA(7-97):

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTTACTTAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAA

U6-DR-spacer-DR (S. pyogenes SF370)

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggttttagagctatgctgttttgaatggtcccaaaacNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgttttagagctatgctgttttgaatggtccca aaac TTTTTTT(lowercase underline = direct repeat; N = guide sequence; bold= terminator)

Chimeric RNA containing+48 tracr RNA (S. pyogenes SF370)

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttgaggctagaaatagcaagttaaaata aggctagtccg TTTTTTT(N = guide sequence; first underline = tracr matesequence; secondunderline = tracr sequence; bold = terminator)

Chimeric RNA containing+54 tracr RNA (S. pyogenes SF370)

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaata aggctagtccgttatcaTTTTTTTT (N = guide sequence; first underline = tracr mate sequence;second underline = tracr sequence; bold = terminator)

Chimeric RNA containing+67 tracr RNA (S. pyogenes SF370)

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtg TTTTTTT (N = guide sequence;firstunderline = tracr mate sequence; second underline = tracr sequence;bold = terminator)

Chimeric RNA containing+85 tracr RNA (S. pyogenes SF370)

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccNNNNNNNNNNNNNNNNNNNNgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc TTTTT TT (N= guidesequence; first underline = tracr mate sequence; second underline= tracr sequence; bold = terminator)

CBh-NLS-SpCas9-NLS

CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGaccggtgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACTTTCTTTTTCTTAGCTTGACCAGCTTTCTTAGTAGCAGCAGGACGCTTTAA (underline= NLS-hSpCas9-NLS)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaa TTTTTT (N = guide sequence;firstunderline = tracr mate sequence; second underline = tracr sequence;bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracrmate sequence; secondunderline = tracr sequence; bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgt TTTTTT (N= guide sequence; first underline = tracr mate sequence;second underline= tracr sequence; bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaa TTTTTT (N = guide sequence;firstunderline = tracr mate sequence; second underline = tracr sequence;bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracrmate sequence; secondunderline = tracr sequence; bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgt TTTTTT (N= guide sequence; first underline = tracr matesequence; second underline= tracr sequence; bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgttattgtactctcaagatttaGAAAtaaatcttgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaa TTTTTT (N = guide sequence; firstunderline = tracr mate sequence; second underline = tracr sequence; bold= terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgttttcgttatttaaTTTTTT (N = guide sequence; first underline = tracrmate sequence; secondunderline = tracr sequence; bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR1 Cas9 (with PAM ofNNAGAAW)

NNNNNNNNNNNNNNNNNNNNgttattgtactctcaGAAAtgcagaagctacaatgataaggcttcatgccgaaatcaacaccctgtcattttatggcagg gtgt TTTTTT (N= guide sequence; first underline = tracr mate sequence;second underline= tracr sequence; bold = terminator)

Example chimeric RNA for S. thermophilus LMD-9 CRISPR3 Cas9 (with PAM ofNGGNG)

NNNNNNNNNNNNNNNNNNNNgttttagagctgtgGAAAcacagcgagttaaaataaggcttagtccgtactcaacttgaaaaggtggcaccgattcggt gt TTTTTT (N = guidesequence; first underline = tracr mate sequence;second underline = tracrsequence; bold = terminator)

Codon-optimized version of Cas9 from S. thermophilus LMD-9 CRISPR3 locus(with an NLS at both 5′ and 3′ ends)

ATGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGACCAAGCCCTACAGCATCGGCCTGGACATCGGCACCAATAGCGTGGGCTGGGCCGTGACCACCGACAACTACAAGGTGCCCAGCAAGAAAATGAAGGTGCTGGGCAACACCTCCAAGAAGTACATCAAGAAAAACCTGCTGGGCGTGCTGCTGTTCGACAGCGGCATTACAGCCGAGGGCAGACGGCTGAAGAGAACCGCCAGACGGCGGTACACCCGGCGGAGAAACAGAATCCTGTATCTGCAAGAGATCTTCAGCACCGAGATGGCTACCCTGGACGACGCCTTCTTCCAGCGGCTGGACGACAGCTTCCTGGTGCCCGACGACAAGCGGGACAGCAAGTACCCCATCTTCGGCAACCTGGTGGAAGAGAAGGCCTACCACGACGAGTTCCCCACCATCTACCACCTGAGAAAGTACCTGGCCGACAGCACCAAGAAGGCCGACCTGAGACTGGTGTATCTGGCCCTGGCCCACATGATCAAGTACCGGGGCCACTTCCTGATCGAGGGCGAGTTCAACAGCAAGAACAACGACATCCAGAAGAACTTCCAGGACTTCCTGGACACCTACAACGCCATCTTCGAGAGCGACCTGTCCCTGGAAAACAGCAAGCAGCTGGAAGAGATCGTGAAGGACAAGATCAGCAAGCTGGAAAAGAAGGACCGCATCCTGAAGCTGTTCCCCGGCGAGAAGAACAGCGGAATCTTCAGCGAGTTTCTGAAGCTGATCGTGGGCAACCAGGCCGACTTCAGAAAGTGCTTCAACCTGGACGAGAAAGCCAGCCTGCACTTCAGCAAAGAGAGCTACGACGAGGACCTGGAAACCCTGCTGGGATATATCGGCGACGACTACAGCGACGTGTTCCTGAAGGCCAAGAAGCTGTACGACGCTATCCTGCTGAGCGGCTTCCTGACCGTGACCGACAACGAGACAGAGGCCCCACTGAGCAGCGCCATGATTAAGCGGTACAACGAGCACAAAGAGGATCTGGCTCTGCTGAAAGAGTACATCCGGAACATCAGCCTGAAAACCTACAATGAGGTGTTCAAGGACGACACCAAGAACGGCTACGCCGGCTACATCGACGGCAAGACCAACCAGGAAGATTTCTATGTGTACCTGAAGAAGCTGCTGGCCGAGTTCGAGGGGGCCGACTACTTTCTGGAAAAAATCGACCGCGAGGATTTCCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCTACCAGATCCATCTGCAGGAAATGCGGGCCATCCTGGACAAGCAGGCCAAGTTCTACCCATTCCTGGCCAAGAACAAAGAGCGGATCGAGAAGATCCTGACCTTCCGCATCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACAGCGATTTTGCCTGGTCCATCCGGAAGCGCAATGAGAAGATCACCCCCTGGAACTTCGAGGACGTGATCGACAAAGAGTCCAGCGCCGAGGCCTTCATCAACCGGATGACCAGCTTCGACCTGTACCTGCCCGAGGAAAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGACATTCAATGTGTATAACGAGCTGACCAAAGTGCGGTTTATCGCCGAGTCTATGCGGGACTACCAGTTCCTGGACTCCAAGCAGAAAAAGGACATCGTGCGGCTGTACTTCAAGGACAAGCGGAAAGTGACCGATAAGGACATCATCGAGTACCTGCACGCCATCTACGGCTACGATGGCATCGAGCTGAAGGGCATCGAGAAGCAGTTCAACTCCAGCCTGAGCACATACCACGACCTGCTGAACATTATCAACGACAAAGAATTTCTGGACGACTCCAGCAACGAGGCCATCATCGAAGAGATCATCCACACCCTGACCATCTTTGAGGACCGCGAGATGATCAAGCAGCGGCTGAGCAAGTTCGAGAACATCTTCGACAAGAGCGTGCTGAAAAAGCTGAGCAGACGGCACTACACCGGCTGGGGCAAGCTGAGCGCCAAGCTGATCAACGGCATCCGGGACGAGAAGTCCGGCAACACAATCCTGGACTACCTGATCGACGACGGCATCAGCAACCGGAACTTCATGCAGCTGATCCACGACGACGCCCTGAGCTTCAAGAAGAAGATCCAGAAGGCCCAGATCATCGGGGACGAGGACAAGGGCAACATCAAAGAAGTCGTGAAGTCCCTGCCCGGCAGCCCCGCCATCAAGAAGGGAATCCTGCAGAGCATCAAGATCGTGGACGAGCTCGTGAAAGTGATGGGCGGCAGAAAGCCCGAGAGCATCGTGGTGGAAATGGCTAGAGAGAACCAGTACACCAATCAGGGCAAGAGCAACAGCCAGCAGAGACTGAAGAGACTGGAAAAGTCCCTGAAAGAGCTGGGCAGCAAGATTCTGAAAGAGAATATCCCTGCCAAGCTGTCCAAGATCGACAACAACGCCCTGCAGAACGACCGGCTGTACCTGTACTACCTGCAGAATGGCAAGGACATGTATACAGGCGACGACCTGGATATCGACCGCCTGAGCAACTACGACATCGACCATATTATCCCCCAGGCCTTCCTGAAAGACAACAGCATTGACAACAAAGTGCTGGTGTCCTCCGCCAGCAACCGCGGCAAGTCCGATGATGTGCCCAGCCTGGAAGTCGTGAAAAAGAGAAAGACCTTCTGGTATCAGCTGCTGAAAAGCAAGCTGATTAGCCAGAGGAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCCCTGAAGATAAGGCCGGCTTCATCCAGAGACAGCTGGTGGAAACCCGGCAGATCACCAAGCACGTGGCCAGACTGCTGGATGAGAAGTTTAACAACAAGAAGGACGAGAACAACCGGGCCGTGCGGACCGTGAAGATCATCACCCTGAAGTCCACCCTGGTGTCCCAGTTCCGGAAGGACTTCGAGCTGTATAAAGTGCGCGAGATCAATGACTTTCACCACGCCCACGACGCCTACCTGAATGCCGTGGTGGCTTCCGCCCTGCTGAAGAAGTACCCTAAGCTGGAACCCGAGTTCGTGTACGGCGACTACCCCAAGTACAACTCCTTCAGAGAGCGGAAGTCCGCCACCGAGAAGGTGTACTTCTACTCCAACATCATGAATATCTTTAAGAAGTCCATCTCCCTGGCCGATGGCAGAGTGATCGAGCGGCCCCTGATCGAAGTGAACGAAGAGACAGGCGAGAGCGTGTGGAACAAAGAAAGCGACCTGGCCACCGTGCGGCGGGTGCTGAGTTATCCTCAAGTGAATGTCGTGAAGAAGGTGGAAGAACAGAACCACGGCCTGGATCGGGGCAAGCCCAAGGGCCTGTTCAACGCCAACCTGTCCAGCAAGCCTAAGCCCAACTCCAACGAGAATCTCGTGGGGGCCAAAGAGTACCTGGACCCTAAGAAGTACGGCGGATACGCCGGCATCTCCAATAGCTTCACCGTGCTCGTGAAGGGCACAATCGAGAAGGGCGCTAAGAAAAAGATCACAAACGTGCTGGAATTTCAGGGGATCTCTATCCTGGACCGGATCAACTACCGGAAGGATAAGCTGAACTTTCTGCTGGAAAAAGGCTACAAGGACATTGAGCTGATTATCGAGCTGCCTAAGTACTCCCTGTTCGAACTGAGCGACGGCTCCAGACGGATGCTGGCCTCCATCCTGTCCACCAACAACAAGCGGGGCGAGATCCACAAGGGAAACCAGATCTTCCTGAGCCAGAAATTTGTGAAACTGCTGTACCACGCCAAGCGGATCTCCAACACCATCAATGAGAACCACCGGAAATACGTGGAAAACCACAAGAAAGAGTTTGAGGAACTGTTCTACTACATCCTGGAGTTCAACGAGAACTATGTGGGAGCCAAGAAGAACGGCAAACTGCTGAACTCCGCCTTCCAGAGCTGGCAGAACCACAGCATCGACGAGCTGTGCAGCTCCTTCATCGGCCCTACCGGCAGCGAGCGGAAGGGACTGTTTGAGCTGACCTCCAGAGGCTCTGCCGCCGACTTTGAGTTCCTGGGAGTGAAGATCCCCCGGTACAGAGACTACACCCCCTCTAGTCTGCTGAAGGACGCCACCCTGATCCACCAGAGCGTGACCGGCCTGTACGAAACCCGGATCGACCTGGCTAAGCTGGGCGAGGGAAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAG AAAAAGAAATAA

Example 5 Optimization of the Guide RNA for Streptococcus pyogenes Cas9(Referred to as SpCas9)

Applicants mutated the tracrRNA and direct repeat sequences, or mutatedthe chimeric guide RNA to enhance the RNAs in cells.

The optimization is based on the observation that there were stretchesof thymines (Ts) in the tracrRNA and guide RNA, which might lead toearly transcription termination by the pol 3 promoter. ThereforeApplicants generated the following optimized sequences. OptimizedtracrRNA and corresponding optimized direct repeat are presented inpairs.

Optimized tracrRNA 1 (mutation underlined):

GGAACCATTCAtAACAGCATAGCAAGTTAtAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT

Optimized direct repeat 1 (mutation underlined):

GTTaTAGAGCTATGCTGTTaTGAATGGTCCCAAAAC

Optimized tracrRNA 2 (mutation underlined):

GGAACCATTCAAtACAGCATAGCAAGTTAAtATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT

Optimized direct repeat 2 (mutation underlined):

GTaTTAGAGCTATGCTGTaTTGAATGGTCCCAAAAC

Applicants also optimized the chimeric guideRNA for optimal activity ineukaryotic cells.

Original guide RNA:

NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT

Optimized chimeric guide RNA sequence 1:

NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTAGAAATAGCAAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTTOptimized chimeric guide RNA sequence 2:

NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGGAAACAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

Optimized chimeric guide RNA sequence 3:

NNNNNNNNNNNNNNNNNNNNGTATTAGAGCTATGCTGTATTGGAAACAATACAGCATAGCAAGTTAATATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

Applicants showed that optimized chimeric guide RNA works better asindicated in FIG. 9. The experiment was conducted by co-transfecting293FT cells with Cas9 and a U6-guide RNA DNA cassette to express one ofthe four RNA forms shown above. The target of the guide RNA is the sametarget site in the human Emx 1 locus: “GTCACCTCCAATGACTAGGG”

Example 6 Optimization of Streptococcus thermophilus LMD-9 CRISPR1 Cas9(referred to as St1Cas9)

Applicants designed guide chimeric RNAs as shown in FIG. 12.

The St1Cas9 guide RNAs can under go the same type of optimization as forSpCas9 guide RNAs, by breaking the stretches of poly thymines (Ts).

Example 7 Improvement of the Cas9 System for In Vivo Application

Applicants conducted a Metagenomic search for a Cas9 with smallmolecular weight. Most Cas9 homologs are fairly large. For example theSpCas9 is around 1368aa long, which is too large to be easily packagedinto viral vectors for delivery. Some of the sequences may have beenmis-annotated and therefore the exact frequency for each length may notnecessarily be accurate. Nevertheless it provides a glimpse atdistribution of Cas9 proteins and suggest that there are shorter Cas9homologs.

Through computational analysis, Applicants found that in the bacterialstrain Campylobacter, there are two Cas9 proteins with less than 1000amino acids. The sequence for one Cas9 from Campylobacter jejuni ispresented below. At this length, CjCas9 can be easily packaged into AAV,lentiviruses, Adenoviruses, and other viral vectors for robust deliveryinto primary cells and in vivo in animal models.

>Campylobacter jejuni Cas9 (CjCas9)

MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKLDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK.

The putative tracrRNA element for this CjCas9 is:

TATAATCTCATAAGAAATTTAAAAAGGGACTAAAATAAAGAGTTTGCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTAAAATT

The Direct Repeat sequence is:

ATTTTACCATAAAGAAATTTAAAAAGGGACTAAAAC

The co-fold structure of the tracrRNA and direct repeat is provided inFIG. 6.

An example of a chimeric guide RNA for CjCas9 is:

NNNNNNNNNNNNNNNNNNNNGUUUUAGUCCCGAAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU

Applicants have also optimized Cas9 guide RNA using in vitro methods.FIG. 18 shows data from the St1Cas9 chimeric guide RNA optimization invitro.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Example 8 Sa sgRNA Optimization

Applicants designed five sgRNA variants for SaCas9 for an optimaltruncated architecture with highest cleavage efficiency. In addition,the native direct repeat:tracr duplex system was tested alongsidesgRNAs. Guides with indicated lengths were co-transfected with SaCas9and tested in HEK 293FT cells for activity. A total of 100 ng sgRNAU6-PCR amplicon (or 50 ng of direct repeat and 50 ng of tracrRNA) and400 ng of SaCas9 plasmid were co-transfected into 200,000 Hepa1-6 mousehepatocytes, and DNA was harvested 72-hours post-transfection forSURVEYOR analysis. The results are shown in FIG. 23.

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While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

What is claimed is:
 1. An engineered, non-naturally occurring ClusteredRegularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPRassociated (Cas) (CRISPR-Cas) vector system comprising one or morevectors comprising: a) a first regulatory element operably linked to oneor more nucleotide sequences encoding one or more CRISPR-Cas systempolynucleotide sequences comprising a guide sequence, a tracr RNA, and atracr mate sequence, wherein the guide sequence hybridizes with one ormore target sequences in polynucleotide loci in a eukaryotic cell, b) asecond regulatory element operably linked to a nucleotide sequenceencoding a Type II Cas9 protein, wherein components (a) and (b) arelocated on same or different vectors of the system, wherein theCRISPR-Cas system comprises two or more nuclear localization signals(NLSs) expressed with the nucleotide sequence encoding the Cas9 protein,whereby the one or more guide sequences target the one or morepolynucleotide loci in a eukaryotic cell and the Cas9 protein cleavesthe one or more polynucleotide loci, whereby the sequence of the one ormore polynucleotide loci is modified.
 2. An engineered, non-naturallyoccurring Type II CRISPR-Cas vector system according to claim 1, whereinthe Cas9 protein is mutated with respect to a corresponding wild typeCas9 protein such that the mutated protein is a nickase that lacks theability to cleave one strand of a target polynucleotide, whereby the oneor more guide sequences target the one or more polynucleotide loci in aeukaryotic cell and the Cas9 protein cleaves only one strand of thepolynucleotide loci, whereby the sequence of the one or morepolynucleotide loci is modified.
 3. The system of claim 1 or 2, whereinthe vectors are viral vectors.
 4. The system of claim 3, wherein theviral vectors are retroviral, lentiviral, adenoviral, adeno-associatedor herpes simplex viral vectors.
 5. The system of any of claims 2-4wherein the Cas9 protein comprises one or more mutations in the RuvC I,RuvC II or RuvC III catalytic domains.
 6. The system of any of claims2-4 wherein the Cas9 protein comprises a mutation selected from thegroup consisting of D10A, H840A, N854A and N863A with reference to theposition numbering of a Streptococcus pyogenes Cas9 (SpCas9) protein. 7.The system of any preceding claim, wherein at least one NLS is at ornear amino-terminus of the Cas9 protein and/or at least one NLS is at ornear carboxy terminus of the Cas9 protein.
 8. The system of claim 7,wherein at least one NLS is at or near amino-terminus of the Cas9protein and at least one NLS is at or near carboxy terminus of the Cas9protein.
 9. The system of any preceding claim, wherein the one or moreCRISPR-Cas system polynucleotide sequences comprise a guide sequencefused to a trans-activating cr (tracr) sequence.
 10. The system of anypreceding claim, wherein the CRISPR-Cas system polynucleotide sequenceis a chimeric RNA comprising the guide sequence, the tracr sequence, anda tracr mate sequence.
 11. The system of any preceding claim, whereinthe eukaryotic cell is a mammalian cell or a human cell.
 12. The systemof any preceding claim, wherein the Cas9 protein is codon optimized forexpression in a eukaryotic cell.
 13. Use of the system of any of claims1 to 12 for genome engineering, provided the use does not comprise aprocess for modifying the germ line genetic identity of human beings,and provided that said use is not a method for treatment of the human oranimal body by surgery or therapy.
 14. The use of claim 13 wherein thegenome engineering comprises modifying a target polynucleotide in aeukaryotic cell, modifying expression of a polynucleotide in aeukaryotic cell, generating a model eukaryotic cell comprising a mutateddisease gene, or knocking out a gene.
 15. The use of claim 13 whereinthe use further comprises repairing said cleaved target polynucleotideby inserting an exogenous template polynucleotide, wherein said repairresults in a mutation comprising an insertion, deletion, or substitutionof one or more nucleotides of said target polynucleotide.
 16. The use ofclaim 13 wherein the use further comprises editing said cleaved targetpolynucleotide by inserting an exogenous template polynucleotide,wherein said edit results in a mutation comprising an insertion,deletion, or substitution of one or more nucleotides of said targetpolynucleotide.
 17. The use of claim 15 or 16 wherein the inserting isby homologous recombination.
 18. Use of the system of any of claims 1 to12 in the production of a non-human transgenic animal or transgenicplant.